Grand scale genome manipulation via chromosome swapping in Escherichia coli programmed by three one megabase chromosomes

In bacterial synthetic biology, whole genome transplantation has been achieved only in mycoplasmas that contain a small genome and are competent for foreign genome uptake. In this study, we developed Escherichia coli strains programmed by three 1-megabase (Mb) chromosomes by splitting the 3-Mb chromosome of a genome-reduced strain. The first split-chromosome retains the original replication origin (oriC) and partitioning (par) system. The second one has an oriC and the par locus from the F plasmid, while the third one has the ori and par locus of the Vibrio tubiashii secondary chromosome. The tripartite-genome cells maintained the rod-shaped form and grew only twice as slowly as their parent, allowing their further genetic engineering. A proportion of these 1-Mb chromosomes were purified as covalently closed supercoiled molecules with a conventional alkaline lysis method and anion exchange columns. Furthermore, the second and third chromosomes could be individually electroporated into competent cells. In contrast, the first split-chromosome was not able to coexist with another chromosome carrying the same origin region. However, it was exchangeable via conjugation between tripartite-genome strains by using different selection markers. We believe that this E. coli-based technology has the potential to greatly accelerate synthetic biology and synthetic genomics.     

Cryopreservation of hairy root cultures of Vinca minor (L.) by encapsulation-dehydration

Hairy root cultures of Vinca minor and Ajuga reptans var. atropurpurea could be cryopreserved when the roots were precultured and encapsulated in 2% (w/v) alginate beads with 0.3 M sucrose and 0.5 M glycerol and dehydrated until the bead weight reached 25% of the initial weight before cooling in liquid nitrogen. Preculture and encapsulation of the roots with abscisic acid was effective in increasing the survival rates. For V. minor root tips moreover a sufficiently high survival rate of more than 70% was attained by eliminating glycerol from the preculture medium and dehydration of beads until 23% of the initial weight was reached instead of 25%.     

Genetic studies on reference strains of mutans streptococci

Twenty four reference strains (serotype a-h) belonging to the mutans group of streptococci were compared for DNA fragment patterns of rDNA after treatment with Hind III. It was shown that Streptococcus cricetus (serotype a), S. rattus (serotype b), and S. downei (serotype h) reveals comparatively homogeneous patterns while S. mutans (serotype c, e and f) exhibits differences between the different serotypes as well as within single serotypes. S. sobrinus had an intermediary diversity. These data support the previous findings that S. mutans is heterogeneous at the serological, biochemical and genetical level.     

Nucleotide 2',3'-cyclic monophosphokinase from actinomycetes

Streptomyces nucleotide 3'-pyrophosphokinase does not only transfer the 5'-beta, gamma-pyrophosphoryl group of ATP, ATP 3'-pyrophosphate or dATP to a variety of nucleotides at the 3'-OH site, but also adds 2',3'-cyclic terminal monophosphate to some suitable nucleotides with the use of diadenosine 5',5'-polyphosphates (n = 3-5). Examples are pA greater than p, ppA greater than p, pG greater than p, CpG greater than p, etc.     

The rare inversion with a P element at the breakpoint maintained in a natural population of Drosophila melanogaster

We found a rare inversion which is superimposed on the In (2R) NS chromosome. It has been maintained for several years in the Raleigh, N. C., USA population and possibly for more than ten years in the southern populations of the USA. Both the breakpoint of the rare inversion and the corresponding site of the In (2R) NS chromosome have P elements and therefore, the rare inversion might be induced by P element activity in a natural population.     

Production of a plasmin inhibitory substance by Scopolia japonica suspension cultures

A potent inhibitory agent against human plasmin, fibrinolytic proteinase, has been found in the extracts of callus tissue of Scopolia japonica. Effects of cultural conditions on cell growth and production of the plasmin inhibitory substance by this cell line in suspension cultures were examined in MurashigeSkoog's medium. More than l.5 mg of the inhibitor, as t-amino cyclohexane carboxylic acid, a synthetic plasmin inhibitor, were observed to accumulate per ml of medium containing 0.83 g of NH₄NO₃ and 7.6 g of KNO₃ per liter as well as suitable levels of growth hormones. Addiction of antibiotics and deformers were examined in preliminary tests for large scale cultivation. Semicontinuous culture on a small scale in a glass cylinder, was also tested and growth rate of 1.29 g/liter/day (by dry wt) was obtained. Plasmin inhibitory activities in the extracts of the results intact plant and in cultured cells of S. japonica were compared and the results indicated that cell suspension culture was superior to extraction the natural plant for inhibitor production.     

Ultrastructural studies on the formation of yolk granules in the statoblast of a fresh-water bryozoan,Pectinatella gelatinosa

The formation of protein-carbohydrate yolk in the statoblast of a fresh-water bryozoan, Pectinatella gelatinosa, was studied by electron microscopy. Two types (I and II) of yolk cells were distinguished. The type I yolk cells are mononucleate and comprise a large majority of the yolk cells. The type II yolk cells are small in number; they become multinucleate by fusion of cells at an early stage of vitellogenesis. In both types of yolk cells, electron-dense granules (dense bodies) are formed in Golgi or condensing vacuoles, which are then called yolk granules. For the formation of yolk granules, the following processes are considered: 1. Yolk protein is synthesized in the rough-surfaced endoplasmic reticulum (RER) of the yolk cells. 2. The synthesized protein condenses in the cisternal space of the RER and is packaged into small oval swellings, which are then released from the RER as small vesicles (Golgi vesicles, 300-600 A in diameter). 3. The small vesicles fuse with one another to form condensing vacuoles, or with pre-existing growing yolk granules. 4. In the matrix of the condensing vacuoles or growing yolk granules, electron-dense fibers are fabricated and then arranged in a paracrystalline pattern to form the dense body. 5. After the dense body reaches its full size, excess membrane is removed and eventually the yolk granules come to mature. Toward the end of vitellogenesis of the yolk cells, the cytoplasmic organelles are ingested by autophagosomes derived from multivesicular bodies and disappear.     

Downregulation of p‐COUMAROYL ESTER 3‐HYDROXYLASE in rice leads to altered cell wall structures and improves biomass saccharification

p‐Coumaroyl ester 3‐hydroxylase (C3′H) is a key enzyme involved in the biosynthesis of lignin, a phenylpropanoid polymer that is the major constituent of secondary cell walls in vascular plants. Although the crucial role of C3′H in lignification and its manipulation to upgrade lignocellulose have been investigated in eudicots, limited information is available in monocotyledonous grass species, despite their potential as biomass feedstocks. Here we address the pronounced impacts of C3′H deficiency on the structure and properties of grass cell walls. C3′H‐knockdown lines generated via RNA interference (RNAi)‐mediated gene silencing, with about 0.5% of the residual expression levels, reached maturity and set seeds. In contrast, C3′H‐knockout rice mutants generated via CRISPR/Cas9‐mediated mutagenesis were severely dwarfed and sterile. Cell wall analysis of the mature C3′H‐knockdown RNAi lines revealed that their lignins were largely enriched in p‐hydroxyphenyl (H) units while being substantially reduced in the normally dominant guaiacyl (G) and syringyl (S) units. Interestingly, however, the enrichment of H units was limited to within the non‐acylated lignin units, with grass‐specific γ‐p‐coumaroylated lignin units remaining apparently unchanged. Suppression of C3′H also resulted in relative augmentation in tricin residues in lignin as well as a substantial reduction in wall cross‐linking ferulates. Collectively, our data demonstrate that C3′H expression is an important determinant not only of lignin content and composition but also of the degree of cell wall cross‐linking. We also demonstrated that C3′H‐suppressed rice displays enhanced biomass saccharification.