Characterization of the promoter controlling Mona/Gads expression in the megakaryocytic lineage

The deletion of a 260-kb segment containing all the coding DNA sequences (CDS) of chromosome 1 of Leishmania major Friedlin strain was performed through homologous recombination during a transfection experiment. This allowed the selection of a mutant clone containing a linear extra chromosome sizing 155 kb (XC155). The structure of XC155 was determined by restriction analysis and DNA cloning and sequencing of the gel-purified chromosome: it is made of a 'mirror' inverted duplication of the 'right' end of chromosome 1a (approximately 25 kb at each end), and in its central part of a complex tandem amplification of the linearized transfection vector containing the hygromycin resistance gene (over approximately 105 kb). No sequence of the coding region of chromosome 1 (including the 1.6-kb 'switch' region) was found. By contrast, XC155 contains two large (approximately 13 kb) clusters of tandemly repeated subtelomeric sequences (272-bp 'satellite' DNA) as well as telomeric hexamer repeats. This extra chromosome was found to be mitotically stable after >150 generations without selective pressure in vitro. Two sequence elements are considered which may have an effect on mitotic stability and participate to centromeric function in this extra chromosome: the amplification of the input vector and the 272-bp 'satellite' DNA bound by telomeric repeats.     

Homo- and heterotypic fibrillin-1 and -2 interactions constitute the basis for the assembly of microfibrils

Fibrillin-1 and fibrillin-2 constitute the backbone of extracellular filaments, called microfibrils. Fibrillin assembly involves complex multistep mechanisms to result in a periodical head-to-tail alignment in microfibrils. Impaired assembly potentially plays a role in the molecular pathogenesis of genetic disorders caused by mutations in fibrillin-1 (Marfan syndrome) and fibrillin-2 (congenital contractural arachnodactyly). Presently, the basic molecular interactions involved in fibrillin assembly are obscure. Here, we have generated recombinant full-length human fibrillin-1, and two overlapping recombinant polypeptides spanning the entire human fibrillin-2 in a mammalian expression system. Characterization by gel electrophoresis, electron microscopy after rotary shadowing, and reactivity with antibodies demonstrated correct folding of these recombinant polypeptides. Analyses of homotypic and heterotypic interaction repertoires showed N- to C-terminal binding of fibrillin-1, and of fibrillin-1 with fibrillin-2. The interactions were of high affinity with dissociation constants in the low nanomolar range. However, the N- and C-terminal fibrillin-2 polypeptides did not interact with each other. These results demonstrate that fibrillins can directly interact in an N- to C-terminal fashion to form homotypic fibrillin-1 or heterotypic fibrillin-1/fibrillin-2 microfibrils. This conclusion was further strengthened by double immunofluorescence labeling of microfibrils. In addition, the binding epitopes as well as the entire fibrillin molecules displayed very stable properties.     

A family of highly diverse human and mouse genes structurally links leukocyte FcR,gp42 and PECAM-1

A group of genes encoding proteins structurally related to the leukocyte Fc receptors (FcRs) and termed the IFGP family was identified in human and mouse. Sequences of four human and two mouse cDNAs predict proteins differing by domain composition. One of the mouse cDNAs encodes a secreted protein, which, in addition to four immunoglobulin (Ig)-like domains, contains a scavenger receptor superfamily-related domain at the C-terminus. The other cDNAs code for the type I transmembrane proteins with the extracellular parts comprised of one to six Ig-like domains. Five homologous types of the Ig-like domains were defined and each protein was found to have a unique combination of the domain types. The cytoplasmic tails of the transmembrane proteins show different patterns of the tyrosine-based signal motifs. While the human IFGP members appear to be B-cell antigens, the mouse genes have a broader tissue distribution with predominant expression in brain. Sequence comparisons revealed that the IFGP family may be regarded as a phylogenetic link joining the leukocyte FcRs with the rat NK cell-specific gp42 antigen and platelet endothelial cell adhesion molecule-1 (PECAM-1), two mammalian leukocyte receptors whose close relatives were not found previously. It is suggested that FcRs, the IFGP proteins and gp42 have arisen by a series of duplications from a common ancestor receptor comprised of five Ig-like domains. The organization of the human genes shows that the IFGP family evolved through differential gain and loss of exons due to recombination and/or mutation accumulation in the duplicated copies.     

Maleimides stimulate oxygen reduction in illuminated thylakoids

N-ethylmaleimide (NEM) and N,N'-(1,4-phenylene)dimaleimide (PDM) were discovered to stimulate light-induced oxygen uptake in isolated thylakoids, and PDM provided the same stimulation at one order less concentrations. Oxygen uptake rate increased promptly after NEM or PDM addition to thylakoids. The inhibitors of photosynthetic electron transport as well as catalase decreased this rate close to zero, whereas ascorbate increased it almost three-fold. Dithiothreitol suppressed oxygen uptake stimulated by NEM. NEM stimulated light-induced reduction of cytochrome c, and this stimulation was suppressed by superoxide dismutase. It was concluded that NEM and PDM being reduced can effectively reduce molecules O(2) producing superoxide radicals.     

Regulation of the ubiquitin-conjugating enzyme hHR6A by CDK-mediated phosphorylation

Cell cycle progression in eukaryotes is mediated by phosphorylation of protein substrates by the cyclin-dependent kinases (CDKs). We screened a cDNA library by solid-phase phosphorylation and isolated hHR6A as a CDK2 substrate. hHR6A is the human homologue of the product of the Saccharomyces cerevisiae RAD6/UBC2 gene, a member of the family of ubiquitin-conjugating enzymes. hHR6A is phosphorylated in vitro by CDK-1 and -2 on Ser120, a residue conserved in all hHR6A homologues, resulting in a 4-fold increase in its ubiquitin-conjugating activity. In vivo, hHR6A phosphorylation peaks during the G2/M phase of cell cycle transition, with a concomitant increase in histone H2B ubiquitylation. Mutation of Ser120 to threonine or alanine abolished hHR6A activity, while mutation to aspartate to mimic phosphorylated serine increased hHR6A activity 3-fold. Genetic complementation studies in S.cerevisiae demonstrated that hHR6A Ser120 is critical for cellular proliferation. This is the first study to demonstrate regulation of UBC function by phosphorylation on a conserved residue and suggests that CDK-mediated phosphorylation of hHR6A is an important regulatory event in the control of cell cycle progression.     

Radical-generating coordination complexes as tools for rapid and effective fragmentation and fluorescent labeling of nucleic acids for microchip hybridization

DNA microchip technology is a rapid, high-throughput method for nucleic acid hybridization reactions. This technology requires random fragmentation and fluorescent labeling of target nucleic acids prior to hybridization. Radical-generating coordination complexes, such as 1,10-phenanthroline-Cu(II) (OP-Cu) and Fe(II)-EDTA (Fe-EDTA), have been commonly used as sequence nonspecific "chemical nucleases" to introduce single-strand breaks in nucleic acids. Here we describe a new method based on these radical-generating complexes for random fragmentation and labeling of both single- and double-stranded forms of RNA and DNA. Nucleic acids labeled with the OP-Cu and the Fe-EDTA protocols revealed high hybridization specificity in hybridization with DNA microchips containing oligonucleotide probes selected for identification of 16S rRNA sequences of the Bacillus group microorganisms.We also demonstrated cDNA- and cRNA-labeling and fragmentation with this method. Both the OP-Cu and Fe-EDTA fragmentation and labeling procedures are quick and inexpensive compared to other commonly used methods. A column-based version of the described method does not require centrifugation and therefore is promising for the automation of sample preparations in DNA microchip technology as well as in other nucleic acid hybridization studies.     

Biochemical characterisation of the 56 and 82 kDa immunodominant gametocyte antigens from Eimeria maxima

Two immunodominant gametocyte antigens from Eimeria maxima with M(r) 56 kDa and M(r) 82 kDa have been identified previously as potential candidates for inclusion in a recombinant subunit vaccine against coccidiosis in poultry. Here, these proteins have been biochemically characterised, immunolocalised within the parasite, and sequences for their amino termini determined. These antigens co-purify by affinity chromatography suggesting an interaction with each other. However, separation of the proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of beta-mercaptoethanol did not reveal the presence of inter-chain disulphide bonds. The true masses of the 56 and 82 kDa antigens are 52450 and 62450 Da, respectively, as determined by mass spectrometry. TX-114 separations suggested that they exist, in part, as soluble proteins within the parasite, and immunolocalisation studies indicated that they were found in the wall forming bodies of macrogametocytes. Separation of the proteins by 2D SDS-PAGE revealed that they are acidic in nature and heterogeneous in charge. Cleavage by neuraminidase and O-glycosidase indicated that the presence of O-linked glycans contributed to some of the charge microheterogeneity of both proteins. The absence of these O-glycans however, did not abolish antibody recognition, suggesting that the development of a recombinant subunit vaccine is possible. A more extensive investigation of the carbohydrate moieties of these proteins revealed that they also possess glucose, fucose, mannose and galactose. There was no evidence for the presence of N-linked glycans. The 56 and 82 kDa antigens were separated from a mixture of proteins in a crude gametocyte lysate by 2D SDS-PAGE, the proteins isolated, and the N-terminus amino acid sequence determined. They showed no homology to each other at the N-terminus, or to any other previously characterised protein. Characterisation of these novel proteins has provided further insights into the molecular mechanisms of gametocyte differentiation in E. maxima.     

Spatial distribution of leaf nitrogen and photosynthetic capacity within the foliage of individual trees: disentangling the effects of local light quality,leaf irradiance,and transpiration

There is presently no consensus about the factor(s) driving photosynthetic acclimation and the intra-canopy distribution of leaf characteristics under natural conditions. The impact was tested of local (i) light quality (red/far red ratio), (ii) leaf irradiance (PPFD(i)), and (iii) transpiration rate (E) on total non-structural carbohydrates per leaf area (TNC(a)), TNC-free leaf mass-to-area ratio (LMA), total leaf nitrogen per leaf area (N(a)), photosynthetic capacity (maximum carboxylation rate and light-saturated electron transport rate), and leaf N partitioning between carboxylation and bioenergetics within the foliage of young walnut trees grown outdoors. Light environment (quantity and quality) was controlled by placing individual branches under neutral or green screens during spring growth, and air vapour pressure deficit (VPD) was prescribed and leaf transpiration and photosynthesis measured at branch level by a branch bag technique. Under similar levels of leaf irradiance, low air vapour pressure deficit decreased transpiration rate but did not influence leaf characteristics. Close linear relationships were detected between leaf irradiance and leaf N(a), LMA or photosynthetic capacity, and low R/FR ratio decreased leaf N(a), LMA and photosynthetic capacity. Irradiance and R/FR also influenced the partitioning of leaf nitrogen into carboxylation and electron light transport. Thus, local light level and quality are the major factors driving photosynthetic acclimation and intra-canopy distribution of leaf characteristics, whereas local transpiration rate is of less importance.     

Peptide nucleic acid (PNA) facilitates multistranded hybrid formation between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded DNA probes

RecA protein-coated single-stranded DNA probes, known as RecA nucleoprotein filaments, bind specifically to homologous DNA sequences within double-stranded DNA targets, forming multistranded probe-target DNA hybrids. This DNA hybridization reaction can be used for sequence-specific gene capture, gene modification, and gene regulation. Thus, factors that enhance the efficiency of the hybridization reaction are of significant practical importance. We show here that the hybridization of a peptide nucleic acid (PNA) within or adjacent to the probe-target homology region significantly enhances the yield of hybrid DNA formed in the reaction between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded (css)DNA probes. The possible mechanisms and the advantages of using RecA nucleoprotein filaments in combination with PNA for genomic DNA cloning and mutagenesis are presented.