Restriction analysis of lambda EMBL3 background recombinants: occurrence of lambda phages carrying a head to tail oriented left arm DNA sequence

Summary Eight representative recombinant background clones of λEMBL3 were analysed usingKpnI,BamHI,SalI,EcoRI andHindIII digestion. We found that λEMBL3 carries its own left arm in theBamHI cloning site. In this way, recombinant molecules were found to be generated which can grow onEscherichia coli strain NM539. In all cases analysed, the left arm DNA was inserted in a head to tail orientation. Seven clones carried a restoredBamHI site at thecos site-BamHI site connection. In the region where the inserted left arm and the right arm were ligated,BamHI cloning produces a large palindromic sequence consisting of two polylinkers. ThisBamHI site was incompletely cleaved in all cases analysed. We assume that a part of the λ DNA molecule in this region shows a cruciform structure prohibiting recognition or cleavage of this site by restriction endonucleaseBamHI.     

An expression vector inhibits gene expression in Xenopus embryos by antisense RNA

Summary An expression vector was constructed containing the entire bovine papilloma virus (BPV-1) genome and part of the a-actin gene of Xenopus laevis cloned in the antisense orientation into the neomycin resistance gene under the control of the herpes simplex virus (HSV) thymidine kinase (TK) promoter. When this vector is microinjected into X. laevis embryos it replicates extrachromosomally, at least up to the tadpole stage, and a fusion RNA is synthesized after the mid blastula transition (MBT). The expression of the antisense gene results in a morphological abnormality of somites demonstrating that antisense RNA generated by an episomal replicating expression vector can inhibit the expression of a selected gene during early embryogenesis of X. laevis.     

Structural analysis of the mitotic cycle in pre-gastrula Xenopus embryos

The long-known phenomenon of karyomere (chromosome vesicle) formation at early telophase of the nuclear cycle during early embryogenesis of a wide range of organisms including amphibians (Rubaschkin 1905; for review, see Richards 1917) was investigated in the early cleavage cycles of Xenopus laevis embryos before the mid blastula transition. Embryos were fixed and Epon embedded at successive time intervals and consecutive thick (3 m) and ultrathin sections cut. Using conventional light microscopy at low magnification as well as phase and/or interference contrast video microscopy at high magnification, a substantial amount of information could be obtained from the analysis of optical sections in thick-sectioned material. In addition, details of the ultrastructural organization could be analysed from corresponding ultrathin sections by electron microscopy. The light microscopic analysis of serial thick sections allowed precise determination of the arrangement and sizes of telophase karyomere structures during the embryonic nuclear division cycle. It was found that small, widely spaced 1st order karyomeres fuse to larger (2nd order) karyomeres which then progressively exhibit lateral fusion of neighbouring karyomeres. The final coalescence of adjacent karyomeres marks the onset of the reorganization of the typical interphase nuclear structure. The data are discussed with regard to the occurrence of karyomeres during the embryonic nuclear cycle of arthropods, dipteran insects, and echinoderms as well as recent progress in the use of Xenopus egg extracts for in vitro assembly of nuclear structures around protein-free DNA.     

Supersensitivity to catecholamines after impairment of extraneuronal uptake or catechol-O-methyl transferase.

In cat papillary muscle and nictitating membrane block of extraneuronal catechol-O-methyl transferase (COMT) by 3',4'-dihydroxy-alpha-methyl propiophenone (U-0521) or of extraneuronal uptake by hydrocortisone causes supersensitivity to catecholamines whenever the experimental conditions result in a high sensitivity of the organ to catecholamines. After block of monoamine oxidase the extraneuronal O-methylation of (-)-[3H]norepinephrine by the isolated nictiating membrane is due to two O-methylating systems (Km and Vmax: 7.5 muM and 0.73 nmoles - g-1 - min-1, and 131 muM and 8.5 nmoles - g-1 - min-1, respectively). Hydrocortisone (28 muM) blocked the activity of the high affinity system without affecting the low affinity system. Apparently, there exists an extraneuronal compartment of high affinity that has a hydrocortisone-sensitive uptake mechnism; this compartment influences the concentration of catecholamines below the Km of this compartment. Supersensitivity ensues when either uptake or enzyme is blocked. Since the sensitivity effects of U-0521 and hydrocortisone are not additive, the high affinity compartment must a) metabolize most of the catecholamine transported into the compartment, and b) have a limited storage capacity for catecholamines after block of COMT.     

DNA-fluorescence of mammalian intra-nucleolar chromatin detected by confocal laser scanning microscopy (CLSM)

The complex spatial DNA distribution in the mammalian interphase nucleus was investigated in Feulgen stained thick sections through mouse trophoblast giant nuclei after Lowicryl embedding. DNA-fluorescence was visualized using confocal laser scanning microscopy. Our results show that the spatial arrangement of major interphase chromatin areas can be precisely documented, including the distribution of small intra-nucleolar chromatin zones.     

Tubulin diversity in trophozoites of<Emphasis Type="Italic"> Giardia lamblia</Emphasis>

Giardia lamblia is a diplomonad that parasitizes the small intestine of vertebrates. The trophozoite is multiflagellar and its cytoskeleton presents a complex organization of microtubular structures. One of these, the adhesive disk, consists of a microtubular spiral. The median body, whose function is not yet determined, is also composed by microtubules. The cell has eight flagella and two microtubule sheets, known as the funis. In this study we used several antibodies and immunofluorescence microscopy to help in the characterization of these structures. We observed that Giardia tubulin reacts with antibodies raised against very distinct immunogens. The antibodies used were against: (1) alpha-tubulin from chicken embryo brain, Trypanosoma brucei, sea urchin sperm, Paramecium, acetylated alpha-tubulin from Paramecium, and tyrosinated alpha-tubulin, (2) beta-tubulin from chicken embryo brain and Physarum polycephalum, and (3) an antibody with specificity to beta-tubulin having as immunogen the FtsZ bacterial protein. Each cytoskeletal structure of Giardia presented a distinct pattern of labeling by the several antibodies used. These data indicate that even being considered one of the most ancient of organisms, Giardia shares similarities (at least in relation to tubulin) with other organisms. They also open some questions about the organization and composition of its microtubular structures.