Retrovirus genomes methylated by mammalian but not bacterial methylase are non-infectious

The biological importance of DNA methylation for gene expression in eukaryotes is becoming increasingly evident, and a direct role of methylation in gene expression has been suggested by an analysis of the infectivity of integrated retroviral genomes in a transfection assay. These studies, however, did not address whether specific methylatable residues are involved in gene regulation. Methylation by sequence-specific bacterial DNA methylases has been shown to suppress the expression of some genes, but not others. To investigate the effect of methylation on gene expression without having to rely on sequence-specific methylases, a rat liver enzyme was used to methylate in vitro all C-G dinucleotides of a proviral genomic clone. This treatment reduced the biological activity of Moloney murine leukaemia virus (M-MuLV) proviral DNA by more than three orders of magnitude, whereas complete methylation of 35 HpaII sites in the same DNA had only a marginal effect. The rat methylase-induced inactivation was reversible, as treatment of recipient cells with 5-azacytidine rendered the non-infectious viral genomes biologically active. This suggests that methylation in other C-G dinucleotides than those detectable with restriction enzymes can be crucial for gene expression.     

Studies on the thyroid in transgenic mice expressing the genes for human and bovine growth hormone

The thyroid glands of transgenic mice (TM) expressing the genes for human (h) and bovine (b) growth hormone (GH) were studied. The percentages of larger follicles in hGH TM of either sex were significantly greater than in the corresponding normal littermates, and follicles ranging up to 350 microns in diameter were present in male hGH TM. In contrast, thyroid follicles were only slightly enlarged in male bGH TM, and were unchanged in female bGH TM. The serum concentrations of T4 were significantly decreased in male bGH TM and not altered in the other groups. Serum concentrations of T3 were slightly, but significantly increased in female hGH TM and female bGH TM, but were unaffected in male TM of either type. Since the principal difference between these foreign GHs in rodents is the additional lactogenic activity of human GH, these results may indicate that the effects of prolactin can influence the development of the thyroid.     

Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells

Long-term reconstitution of the lymphohaematopoietic cells of a mouse after lethal irradiation requires the transplantation of at least (5-10) x 10(3) bone marrow cells. Several cell-separation techniques based on cell-surface characteristics have been used in attempts to identify the pluripotent haematopoietic stem cells (PHSC), and have allowed the long-term engraftment of lethally irradiated mice with an enriched fraction of fewer than 200 marrow cells. But these techniques enrich not only for PHSC but also for haematopoietic progenitors, especially day-12 spleen colony-forming units (CFU-S). Although day-12 CFU-S have been postulated to be primitive multipotential haematopoietic progenitors, with day-8 CFU-S representing later, more committed progenitors, recent evidence suggests that neither of these CFU-S represents mouse PHSC. Here we report that counterflow centrifugal elutriation, which sorts cells on the basis of size and density, can separate PHSC from these less primitive progenitors. The fraction containing the largest cells was enriched for the granulocyte-macrophage colony-forming units (CFU-GM), but gave only transient, early engraftment and was therefore depleted of PHSC. The intermediate fraction was enriched for CFU-S, but depleted of CFU-GM. Despite being devoid of CFU-GM and CFU-S, the fraction consisting of only morphological lymphocytes gave sustained, albeit delayed, reconstitution of all lymphohaematopoietic cells, and was therefore enriched for PHSC. We conclude that there are two vital classes of engrafting cells: committed progenitors, which provide initial, unsustained engraftment, and PHSC, which produce delayed, but durable, engraftment. Therefore for late haematological reconstitution, PHSC must be transplanted with a distinguishable source of early engrafting cells, thereby allowing the lethally irradiated host to survive initial aplasia.     

Untersuchungen über die β-adrenolytische Wirkung sowie den Einfluss auf Refraktärzeit und Kontraktilität von INPEA und MJ1999 am Meerschweinchenvorhof

Summary In isolated guinea-pig atria, the-adrenolytic isomerd(–)INPEA provoked a stronger prolongation of the refractory period (rp) than did the inactivel(+) isomer. The-adrenolytic drug MJ 1999 prolonged likewise the rp, although it caused no unspecific effect, i.e. an inhibition of contractile force. These observations lead to the conclusion that not only the unspecific local anaesthetic but also the specific-adrenolytic effect is of importance for the prolongation of rp.     

Levels ofα 1 in the spring and autumn seasons

Summary In a group of 84 pairs of 11-year-old children of both sexes, the level of the ₁-antitrypsin ( ₁-AT) were ascertained in the autumn and spring. Although the mean levels of ₁-AT in the two seasons hardly differed, the highly significant seasonal changes in the distribution curves of ₁-AT values were noted in boys, whereas the levels showed higher stability in girls.     

Structural basis for the selectivity of the external thioesterase of the surfactin synthetase

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, fungi and plants use two different types of thioesterases for the production of highly active biological compounds. Type I thioesterases (TEI) catalyse the release step from the assembly line of the final product where it is transported from one reaction centre to the next as a thioester linked to a 4'-phosphopantetheine (4'-PP) cofactor that is covalently attached to thiolation (T) domains. The second enzyme involved in the synthesis of these secondary metabolites, the type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4'-PP cofactors of holo-T domains of NRPS and PKS systems. Mispriming of 4'-PP cofactors by acetyl- and short-chain acyl-residues interrupts the biosynthetic system. This repair reaction is very important, because roughly 80% of CoA, the precursor of the 4'-PP cofactor, is acetylated in bacteria. Here we report the three-dimensional structure of a type II thioesterase from Bacillus subtilis free and in complex with a T domain. Comparison with structures of TEI enzymes shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. Furthermore, we show that the TEII enzyme exists in several conformations of which only one is selected on interaction with its native substrate, a modified holo-T domain.     

Dynamic thiolation-thioesterase structure of a non-ribosomal peptide synthetase

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties. Like fatty acid synthases (FAS), these enzymes are organized in modular assembly lines in which each module, made of conserved domains, incorporates a given monomer unit into the growing chain. Knowledge about domain or module interactions may enable reengineering of this assembly line enzymatic organization and open avenues for the design of new bioactive compounds with improved therapeutic properties. So far, little structural information has been available on how the domains interact and communicate. This may be because of inherent interdomain mobility hindering crystallization, or because crystallized molecules may not represent the active domain orientations. In solution, the large size and internal dynamics of multidomain fragments (>35 kilodaltons) make structure determination by nuclear magnetic resonance a challenge and require advanced technologies. Here we present the solution structure of the apo-thiolation-thioesterase (T-TE) di-domain fragment of the Escherichia coli enterobactin synthetase EntF NRPS subunit. In the holoenzyme, the T domain carries the growing chain tethered to a 4'-phosphopantetheine whereas the TE domain catalyses hydrolysis and cyclization of the iron chelator enterobactin. The T-TE di-domain forms a compact but dynamic structure with a well-defined domain interface; the two active sites are at a suitable distance for substrate transfer from T to TE. We observe extensive interdomain and intradomain motions for well-defined regions and show that these are modulated by interactions with proteins that participate in the biosynthesis. The T-TE interaction described here provides a model for NRPS, PKS and FAS function in general as T-TE-like di-domains typically catalyse the last step in numerous assembly-line chain-termination machineries.