Alternative mating strategies in Atlantic salmon and brown trout

By screening variable number of tandem repeat (VNTR) loci, multiple paternity within clutches has been found in wild populations of southern European Atlantic salmon (Salmo salar) and brown trout (Salmo trutta). For Atlantic salmon, we determined the relative contribution of alternative male phenotypes to the next generation. Individual males that are morphologically juvenile yet sexually mature fertilized a large proportion of eggs, and they thereby contributed to an increase of genetic variability in wild populations via (1) balancing the sex ratio, (2) increasing outbreeding, and (3) enlarging the effective population size, in part a consequence of (1) and (2). In addition, these precocious males ensured that interspecific spawns involving Atlantic salmon females and brown trout males (a fairly common occurrence in southern Europe where the two species are sympatric) resulted mostly in Atlantic salmon progeny. For brown trout, preliminary genetic results indicated that multiple paternity, when present, was not due to alternative mating strategies by males, but rather to successive fertilizations by adult suitors.     

The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation

A MADS box gene, FLF (for FLOWERING LOCUS F ), isolated from a late-flowering, T-DNA-tagged Arabidopsis mutant, is a semidominant gene encoding a repressor of flowering. The FLF gene appears to integrate the vernalization-dependent and autonomous flowering pathways because its expression is regulated by genes in both pathways. The level of FLF mRNA is downregulated by vernalization and by a decrease in genomic DNA methylation, which is consistent with our previous suggestion that vernalization acts to induce flowering through changes in gene activity that are mediated through a reduction in DNA methylation. The flf-1 mutant requires a greater than normal amount of an exogenous gibberellin (GA3) to decrease flowering time compared with the wild type or with vernalization-responsive late-flowering mutants, suggesting that the FLF gene product may block the promotion of flowering by GAs. FLF maps to a region on chromosome 5 near the FLOWERING LOCUS C gene, which is a semidominant repressor of flowering in late-flowering ecotypes of Arabidopsis.     

Interactions of surface-confined DNA with electroreduced mitomycin C comparison with acid-activated mitomycin C

The anticancer activity of the antineoplastic drug mitomycin C (MC) was investigated using transfer stripping cyclic voltammetry (TSCV) with single-stranded DNA-modified hanging mercury drop electrode (HMDE). Reductive activation of MC is necessary for drug covalent binding to DNA, and we have found that some potential-controlled interactions of MC with DNA occur at the electrode, i.e. MC can be activated by electroreduction. Acid and electroreductive MC activations were compared and different adducts were subsequently generated, suggesting that the drug can bind to DNA in more than one way. Under conditions of acid activated MC, a monofunctional adduct between C-1 of MC and N-7 of guanine was formed on the electrode surface, reduced at - 0.44 V (vs. SCE). However, when the DNA-modified electrode was immersed in a MC solution and potentials corresponding to the quinone moiety reduction (- 0.3 V or more negative vs. SCE) were applied, an intrastrand bifunctional adduct between C-1 and C-10 of MC and two N-7 of a pair of adjacent guanines in ssDNA were formed at the electrode, reduced at - 0.49 V, i.e. 50 mV more negative than the monoadduct. The results presented in this paper show for the first time electrochemical detection of DNA-MC adducts at the hanging mercury drop electrode.     

Mammalian artificial chromosome pilot production facility: large-scale isolation of functional satellite DNA-based artificial chromosomes.

BACKGROUND: A pilot production facility has been established to isolate mammillian artificial chromosomes at high purity by using flow cytometric techniques. Dicentric chromosomes have been generated by the targeted amplification of pericentric heterochromatic and centromeric DNA by activating the "megareplicator." Breakage of these dicentric chromosomes generates satellite DNA-based artificial chromosomes (SATAC) from 60 to 400 megabases. METHODS: For large-scale production, we have developed cell lines capable of carrying one or two SATACs. A SATAC, because of a high adenine-thymine (AT) composition, is easily identified and sorted by using chromomycin A3 and Hoechst 33258 stains and a dual laser high-speed flow cytometer. A prototype SATAC (60 megabases) has been characterized. The prototype SATAC has been isolated from an original rodent/human hybrid cell line and transferred by using modified microcell fusion into a CHO production cell line. RESULTS: Metaphase chromosomes from this production cell line were isolated in a modified polyamine buffer, stained, and sorted by using a modified sheath buffer that maintains condensed chromosomes. SATACs are routinely sorted at rates greater than 1 million per hour. Sorted SATACs have been transferred to a variety of cells by using microcell fusion technology and were found to be functional. CONCLUSIONS: By developing new SATAC containing cell lines with fewer numbers of chromosomes in conjunction with operating a high speed flow sorter we have effectively generated an efficient production facility geared purely for the isolation of SATACs.     

A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy

Mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure. In order to extend the utility of ACEs, we have established the ACE System, a versatile and flexible platform for the reliable engineering of ACEs. The ACE System includes a Platform ACE, containing >50 recombination acceptor sites, that can carry single or multiple copies of genes of interest using specially designed targeting vectors (ATV) and a site-specific integrase (ACE Integrase). Using this approach, specific loading of one or two gene targets has been achieved in LMTK⁻ and CHO cells. The use of the ACE System for biological engineering of eukaryotic cells, including mammalian cells, with applications in biopharmaceutical production, transgenesis and gene-based cell therapy is discussed.     

Small marker chromosomes in two patients with segmental aneusomy for proximal 17p

We report a nine-year-old girl (patient 1934) and a five-year-old boy (patient 2170) with small, de novo supernumerary marker chromosomes (SMCs) derived from proximal 17p. The clinical features of patient 1934 include developmental delay, triangular face, prominent forehead, low set ears, dental abnormalities, a high arched palate, long, flexible fingers, and joint laxity. Patient 2170 is affected with developmental delay, oral-motor dyspraxia/verbal apraxia, thick upper and lower lips, bilateral fifth finger clinodactyly, joint laxity and mild hypotonia. G-banded chromosome analysis of patient 1934 revealed mosaicism for a SMC in 72% of peripheral lymphocytes analyzed, whereas analysis of patient 2170 identified a smaller SMC present in 100% of cells analyzed. Fluorescence in situ hybridization (FISH) studies demonstrated that both of the SMCs derived from 17p10-p11.2. Using FISH and array-CGH analysis, the proximal breakpoints mapped within the centromere and the distal breakpoints were both located within the Smith-Magenis syndrome (SMS) common deletion region. We compare the clinical characteristics of our patients with those previously reported to have either SMC including 17p or duplications of proximal 17p in an effort to further delineate the phenotype of trisomy 17p10-p11.2 and to elucidate genotype-phenotype correlations.     

Frictional properties of contacting surfaces in the hemelytra-hindwing locking mechanism in the bug<Emphasis Type="Italic"> Coreus marginatus</Emphasis> (Heteroptera,Coreidae)

The structure and function of the hemelytra-to-hindwing locking mechanism of the bug Coreus marginatus were analysed. The system consists of a cuticular protrusion in the ventral side of the hemelytra, which locks the subcostal border of the hindwing in flight. The speed and distance slid by both surfaces against one another during flight were assessed using a combination of high-speed video recordings and a 2D geometrical model. The friction coefficient between sliding surfaces was assessed using a micromanipulator, coupled with force transducers. This was done under three experimental conditions: freshly dissected, air dried and rehydrated ethanol preserved samples. The results showed a high speed of sliding, approximately 0.18 m s^–1, with a relatively low friction coefficient (0.2 ). There was no evident difference in the friction measured under the various treatments, with the exception of the rehydrated condition, which was lower. The surface morphology of the wing locking mechanism, namely outgrowths of one part having rounded edges, and completely flat surface on the counterpart, effectively aids in the reduction of friction at the microscopic level. The structure is effective even dry, and after being preserved in ethanol, suggesting that no cuticle secreted lubrication substance is responsible for its effectiveness. The ultrastructure presumably confers mechanical stability to the system under the high load it is subjected to in flight.