Genetic determination of NOR activity in human lymphocytes from twins

Summary Activity of nucleolar organizer regions (NORs) was studied in cultured blood lymphocytes from 20 monozygotic (MZ) and 20 dizygotic (DZ) twin pairs. The number of Agstained NORs, the degree of staining, and the frequency of acrocentric associations were used as criteria of the NOR activity, the acrocentric chromosomes being identified by G-banding. Analysis of intrapair concordance as well as of intrapair variance showed the number of Ag+NORs and the size of Ag-deposits to be highly heritable traits. Intrapair differences in acrocentric association frequency were not significantly higher in DZ compared with MZ twins.     

Sister chromatid differential staining and NOR activity of BrdU-resistant sublines

Summary Two 30 g/ml BrdU-resistant sublines and two 60 g/ml BrdU-resistant sublines are induced from a Chinese hamster cell line Wg3h (HGPRT^–) by one-step and two-step selections, respectively. By inoculating the cells into BrdU-free medium or by adding more BrdU into the culture medium for 26–27 h, it was found that the two BrdU-resistant sublines analysed have very clear sister chromatid differential (SCD) staining patterns. This indicates that some of the nuclear DNA of the BrdU-resistant cells incorporate with BrdU to reach a kinetic balance. Frequencies of sister chromatid exchange (SCE) of the resistant cells are twice to four times as high as those of the Wg3h cells, depending on which BrdU-resistant subline is analysed. The SCE frequencies of the resistant cells also increase with the BrdU concentration in the medium. Analysis of silver-stained nucleolar organizer regions (NORs) indicates that the NOR activity of three out of the four BrdU-resistant sublines is significantly suppressed, i.e., averages of the Ag-NOR number and number of the chromosomes bearing Ag-NORs per cell decrease significantly. The degree of suppression for different BrdU-resistant sublines may be quite different. The suppressed NOR activity of the resistant cells can gradually be restored when the cells are inoculated into BrdU-free medium, but the recovery speed is far lower than that of the Wg3h cells. The suppression of the NOR activity of the BrdU-resistant sublines should be due to BrdU toxicity.     

Cyclic nucleotide phosphodiesterase activity in barley seeds

Barley seeds (Hordeum vulgare L. cv. Himalaya) contain an enzymatic activity which catalyzes the hydrolysis of adenosine cyclic 3′: 5′-monophosphate and adenosine cyclic 2′: 3′-monophosphate. A large portion of the enzymatic activity is present in the dry seed, existing in both soluble and particulate form. Secretion of the soluble phosphodiesterase from embryoless seeds is enhanced by gibberellic acid and inhibited by abscisic acid, dinitrophenol, and cycloheximide. Attempts to isolate or detect a phosphodiesterase which specifically hydrolyzes adenosine cyclic 3′: 5′-monophosphate were unsuccessful. Inhibition experiments indicate that probably one enzyme is involved in the hydrolysis of both of these substrates.     

Cisgenic barley with improved phytase activity

The cisgenesis concept implies that plants are transformed only with their own genetic materials or genetic materials from closely related species capable of sexual hybridization. Furthermore, foreign sequences such as selection genes and vector-backbone sequences should be absent. We used a barley phytase gene (HvPAPhy_a) expressed during grain filling to evaluate the cisgenesis concept in barley. The marker gene elimination method was used to obtain marker-free plant lines. Here, the gene of interest and the selection gene are flanked by their own T-DNA borders to allow unlinked integration of the two genes. We analysed the transformants for co-transformation efficiency, increased phytase activities in the grain, integration of the kanamycin resistance gene of the vector-backbone and segregation between the HvPAPhy_a insert and the hygromycin resistance gene. The frequencies of the four parameters imply that it should be possible to select 11 potentially cisgenic T(1) -lines out of the 72 T(0) -lines obtained, indicating that the generation of cisgenic barley is possible at reasonable frequencies with present methods. We selected two potential cisgenic lines with a single extra copy of the HvPAPhy_a insert for further analysis. Seeds from plants homozygous for the insert showed 2.6- and 2.8-fold increases in phytase activities and the activity levels were stable over the three generations analysed. In one of the selected lines, the flanking sequences from both the left and right T-DNA borders were analysed. These sequences confirmed the absence of truncated vector-backbone sequences linked to the borders. The described line should therefore be classified as cisgenic.     

Changes in NOR activity pattern in the presence of supernumerary heterochromatin in the grasshopper Eyprepocnemis plorans

Nucleolus organizer region (NOR) activity was analysed in four types of males of the grasshopper Eyprepocnemis plorans, possessing two kinds of supernumerary heterochromatin: a B chromosome and a supernumerary chromosome segment proximally located on the smallest autosome (S11). In males lacking extra heterochromatin, the four active NORs located on the S9, S10, S11, and X chromosomes showed independent activity patterns, but several kinds of dependence appeared in the presence of supernumerary heterochromatin. Furthermore, temporal changes in NOR activity were observed during the first 2 weeks of adult life in standard males but not in males carrying supernumerary heterochromatin. It is suggested that all these effects are related to the DNA content of both types of extra heterochromatin.     

NOR associations with heterochromatin

Associations between nucleolus organizer regions (NORs) and non-acrocentric chromosomes were scored in 2,800 metaphase spreads from PHA-stimulated lymphocyte cultures (48 h) from 14 individuals. The preparations were both silver stained and C-banded. In order to calculate the expected values for associations, the ratio of heterochromatin length to euchromatin length was established for each subject. Individual C-band lengths and centromeric lengths were also determined. When silver connective (SC) associations with heterochromatin were compared to SC associations with euchromatin, the number of associations with heterochromatin was significantly greater than expected (P less than 0.000001) for each subject. The SC associations were not distributed randomly over the heterochromatin of the non-acrocentrics. Chromosomes 1 and 2 had significantly more than expected. Chromosomes 17, 18, 19, 20, and the Y had fewer than expected. NOR associations with euchromatic segments also showed a nonrandom pattern of distribution.     

The Eucalypt Lignotuber: a Position-dependent Organ

Observational and experimental evidence is presented to showthat the capacity to form a lignotuber is not restricted tothe cotyledonary, and a few succeeding, nodes on the primaryseedling stems of certain eucalypts, but is also a propertyof shoots arising from their accessory buds. Erect secondaryshoots from the lignotuber, may bear lignotubers at a few nodes,rhizostolons at many nodes along their length, and, like stolons,rhizomes may give rise to leafy shoots bearing basal lignotubers.These facts describe a so-called position effect for the productionof lignotubers, in the sense that the term has been used forother position-dependent phenomena in plants. Seedlings of lignotuberousspecies remain competent to form lignotubers over a long periodof adversity during which lignotubers are not formed. Shootsof non-lignotuberous species do not inhibit lignotuber developmentwhen grafted to lignotuberous species. eucalypts, lignotubers, position effect, vegetative propagation, tissue culture, rhizostolons, rhizomes     

Position-dependent motif characterization using non-negative matrix factorization

MOTIVATION: Cis-acting regulatory elements are frequently constrained by both sequence content and positioning relative to a functional site, such as a splice or polyadenylation site. We describe an approach to regulatory motif analysis based on non-negative matrix factorization (NMF). Whereas existing pattern recognition algorithms commonly focus primarily on sequence content, our method simultaneously characterizes both positioning and sequence content of putative motifs. RESULTS: Tests on artificially generated sequences show that NMF can faithfully reproduce both positioning and content of test motifs. We show how the variation of the residual sum of squares can be used to give a robust estimate of the number of motifs or patterns in a sequence set. Our analysis distinguishes multiple motifs with significant overlap in sequence content and/or positioning. Finally, we demonstrate the use of the NMF approach through characterization of biologically interesting datasets. Specifically, an analysis of mRNA 3'-processing (cleavage and polyadenylation) sites from a broad range of higher eukaryotes reveals a conserved core pattern of three elements.     

Silver staining of human acrocentrics in metaphase does not reflect an induced inhibition in NOR activity

Cytological staining with silver nitrate was used in order to study the activity of the nucleolar organizer regions (NORs) in metaphase figures from human lymphocytes exposed to mercury chloride and actinomycin D. The cells were exposed to both compounds either during G1-early S phase, allowing recovery after the exposure, or from G1 until harvest; no recovery was thus allowed in the latter case. HgCl₂ as well as actinomycin D did not influence the silver staining of the acrocentric chromosomes on metaphases. As actinomycin D is known to be an inhibitor of rRNA, as for example confirmed by inhibition of silver staining on interphase cells, our results on metaphase chromosomes indicate that AgNO₃ precipitation, although being a good indicator for nucleolar activation, is not adequate in case of inactivation.     

Partial translocation of NOR and its activity in a balanced carrier and in her cri-du-chat fetus

Summary Cytogenetic analysis of a balanced reciprocal translocation t(5;13) carrier and her unbalanced 5p-conceptus was carried out. This carrier mother had previously given birth to a child with cri-du-chat syndrome. Silver staining demonstrated the breakpoint on 13p within the nucleolus organizer region (NOR). In the carrier, the NORs both at the original site 13p0 and at the translocated site (5p) were silver-stained, indicating that the rRNA genes at both sites were transcribed. The NOR at the derivative chromosome 5 was also silver-stained in the fetus.     

Localization and activity of various peptidases in germinating barley

Summary Germinating barley grains contain at least eight different peptidases: three carboxypeptidase (pH optima 4.8, 5.2, and 5.7), three aminopeptidases which act on aminoacyl--naphthylamides (pH opitima in the hydrolysis of di- and tripeptides at pH 5.8–6.5), and two peptidases which hydrolyse Ala-Gly and Leu-Tyr optimally at pH 7.8 and 8.6 respectively. We have determined the activities of these enzymes in the different tissues of non-germinated grains and followed the changes in the activities during a 5-day germination at 16°C.The aleurone layers contain high activities of all three groups of peptidases; there are no changes in the activities of the five aminopeptidases on germination, while the carboxypeptidases exhibit a small increase of activity. The starchy endosperms contain high carboxypeptidase activities, which increase during germination, but are totally devoid of the five aminopeptidases.All the peptidases exhibit high activities in the scutella; the carboxypeptidases and the enzymes acting on Ala-Gly and Leu-Tyr increase in activity during germination, while the naphthylamidase activities remain constant.The three peptidase groups occur in the seedling as well, but compared to the other tissues the carboxypeptidase activities are very small and the naphthylamidase activities are very high. The last-named enzymes seem to be characteristic for growing tissues.The starchy endosperm contains about two thirds of the total reserve proteins of the grain. Its internal pH during germination is 5.0–5.2, a value at which all the carboxypeptidases are highly active. As these enzymes are present in high concentrations in this tissue, it is probable that they have a central role in the mobilization of the reserve proteins during germination. The high peptidase activities of the scutellum, on the other hand, suggest that some of the hydrolysis products are absorbed as peptides and these are further hydrolysed to amino acids in this tissue.Abbreviations used DTT dithiothreitol - GA₃ gibberellic acid - -NA -naphthylamide - TNBS 2,4,6-trinitrobenzene sulphonic acid - Z- N-carbobenzoxy     

Circadian rhythmicity of nitrate reductase activity in barley leaves

Nitrate reductase (EC 1.6.6.1) activity showed circadian rhythmicity in the first leaf of 8–11 days old barley ( Hordeum vulgare L. cv. Herta) plants. Circadian rhythms were found using both the in vitro and in vivo method for testing the enzyme activity. When the light intensity was reduced from 65 to 20 W m⁻², the amplitude was smaller and the oscillations were damped sooner. In continuous darkness nitrate reductase activity decreased in a two step process. Three different light qualities were tested which all gave the same results.     

Development patterns of telomerase activity in barley and maize

Eukaryotic chromosomes terminate with specialized structures called telomeres. Maintenance of chromosomal ends in most eukaryotes studied to date requires a specialized enzyme, telomerase. Telomerase has been shown to be developmentally regulated in man and a few other multicellular organisms, while it is constitutively expressed in unicellular eukaryotes. Recently, we demonstrated telomerase activity in plant extracts using the PCR-based TRAP (Telomeric Repeat Amplification Protocol) assay developed for human cells. Here we report telomerase activities in two grass species, barley and maize, using a modified, semi-quantitative TRAP assay. Telomerase was highly active in very young immature embryos and gradually declined during embryo development. The endosperm telomerase activity was detectable, but significantly lower than in the embryo and declined during kernel development with no detectable activity in later stages. Telomerase activity in dissected maize embryo axis was several orders of magnitude higher than in the scutellum. Telomerase activity was not detected in a range of differentiated tissues including those with active meristems such as root tips as well as the internode and leaf base. The role of telomerase repression during differentiation and the relationship between chromosome healing and telomerase activity is discussed.     

Position-dependent effects on stability in tricyclo-DNA modified oligonucleotide duplexes

A series of oligodeoxyribonucleotides and oligoribonucleotides containing single and multiple tricyclo(tc)-nucleosides in various arrangements were prepared and the thermal and thermodynamic transition profiles of duplexes with complementary DNA and RNA evaluated. Tc-residues aligned in a non-continuous fashion in an RNA strand significantly decrease affinity to complementary RNA and DNA, mostly as a consequence of a loss of pairing enthalpy ΔH. Arranging the tc-residues in a continuous fashion rescues T_m and leads to higher DNA and RNA affinity. Substitution of oligodeoxyribonucleotides in the same way causes much less differences in T_m when paired to complementary DNA and leads to substantial increases in T_m when paired to complementary RNA. CD-spectroscopic investigations in combination with molecular dynamics simulations of duplexes with single modifications show that tc-residues in the RNA backbone distinctly influence the conformation of the neighboring nucleotides forcing them into higher energy conformations, while tc-residues in the DNA backbone seem to have negligible influence on the nearest neighbor conformations. These results rationalize the observed affinity differences and are of relevance for the design of tc-DNA containing oligonucleotides for applications in antisense or RNAi therapy.