The Codon Usage in the Minimal Natural Cell

A statistical analysis of the variation in contents with the size of the current known smallest genomes, N. deltocephalinicola, C. ruddii, N. equitans, and M. genitalium, enabled the indication of a minimal set of codons capable of naturally building a modern-type free-living unicellular organism in an early stage of evolution. Using a linear regression model, the potential codon distribution in the minimal natural cell was predicted and compared to the composition of the smallest synthetic, JCVI-Syn3.0. The distribution of the molecular weight of potentially coded amino acids was also calculated. The main differences in the features of the minimal natural cell and H. Sapiens genome were analyzed. In this regard, the content percentage of respective amino acids and their polarization charge properties were reported and compared. The fractions of occurring nucleotides were calculated, too. Then, the estimated numbers of codons in a minimal natural cell were related to the expected numbers for random distribution. Shown increase, or decrease in the contents, relative to the calculated random filling was related to the evolutionary preferences, varying with the subsequent eras of the evolution of genetic code.

     

The use of compensatory base change analysis of ITS2 as a tool in the phylogeny of Mucorales, illustrated by the Mucor circinelloides complex

Compensatory base changes (CBCs) in helix II of rDNA ITS2, suggested as a molecular classifier for fungi, were analyzed in Mucor circinelloides and its varieties. Only a few CBCs were found in the complex. Three out of the four accepted formae (f. circinelloides, f. lusitanicus, f. janssenii) did not exhibit CBCs. One CBC was found between strains that form zygospores; consequently, CBC is not always concordant with mating experiments. Strains with two CBC are unable to breed. It is suggested that some strains of the M. circinelloides complex are at the beginning of speciation.     

Optimized perfusion‐based CUBIC protocol for the efficient whole‐body clearing and imaging of rat organs

Whole‐organ and whole‐body optical tissue clearing methods allowing imaging in 3 dimensions are an area of profound research interest. Originally developed to study nervous tissue, they have been successfully applied to all murine organs, yet clearing and imaging of rat peripheral organs is less advanced. Here, a modification of CUBIC clearing protocol is presented. It provides a rapid and simple approach to clear the entire adult rat organism and thus all organs within as little as 4 days. Upgraded perfusion‐based rat CUBIC protocol preserves both anatomical structure of organs and signal from proteinaceous fluorophores, and furthermore is compatible with antibody staining. Finally, it enables also volumetric cells analyses and is tailored for staining of calcium deposits within unsectioned soft tissues.      

Floral nectary and osmophore of <Emphasis Type="Italic">Epipactis helleborine</Emphasis> (L.) Crantz (Orchidaceae)

The analysis of flowers collected at different stages of anthesis provides strong evidence to conclude that the shell-shaped hypochile and the knobs of epichile form a nectary. The scent comes from the aromatic constituents of nectar and the epichile tissue and the apices of all tepals (osmophores). The comparison between pollinated and unpollinated flowers revealed that the anthesis of unpollinated flowers lasted up to the 16th day. The nectariferous secretory cells formed single-layered epidermis and several layers of underlying parenchyma built by small, isodiametric cells with thin walls and dense cytoplasm, relatively large nuclei, supplied by collateral vascular bundles. During the floral lifespan, the residues of secreted material were higher on the hypochile cells. The lipoid-carbohydrate material and lipid globules in the cell walls and in the cytoplasm were localised. The abundance of starch grains was observed at the beginning of anthesis and their gradual reduction during the flower lifespan. At the end of anthesis in unpollinated flowers, the lipoid-carbohydrate-phenolic materials have been demonstrated. The phenolic material was the same as in plastoglobuli. The features such as irregular plasmalemma, the secretory vesicles that fuse with it, fully developed dictyosomes, numerous profiles of ER indicate vesicle-mediated process of secretion. The substances could be transported by vesicles to the periplasmic space via granulocrine secretion and then to the external surface. Both micro-channels and slightly developed periplasmic space were visible in the hypochile epidermis. This is the first time for anatomical survey of secretory tissue in pollinated and unpollinated flowers of E. helleborine.     

Aerobactin biosynthesis and transport genes of plasmid ColV-K30 in Escherichia coli K-12.

The iron-regulated aerobactin operon, about 8 kilobase pairs in size, of the Escherichia coli plasmid ColV-K30 was shown by deletion and subcloning analyses to consist of at least five genes for synthesis (iuc, iron uptake chelate) and transport (iut, iron uptake transport) of the siderophore. The gene order iucABCD iutA was established. The genes were mapped within restriction nuclease fragments of a cloned 16.3-kilobase-pair HindIII fragment. Stepwise deletion and subsequent minicell analysis of the resulting plasmids allowed assignment of four of the five genes to polypeptides of molecular masses 63,000, 33,000 53,000, and 74,000 daltons, respectively. The 74-kilodalton protein, the product of gene iutA, is the outer membrane receptor for ferric aerobactin, whereas the remaining three proteins are involved in biosynthesis of aerobactin. The 33-kilodalton protein, the product of gene iucB, was identified as N epsilon-hydroxylysine:acetyl coenzyme A N epsilon-transacetylase (acetylase) by comparison of enzyme activity in extracts from various deletion mutants. The 53-kilodalton protein, the product of gene iucD, is required for oxygenation of lysine. The 63-kilodalton protein, the product of gene iucA, is assigned to the first step of the aerobactin synthetase reaction. The product of gene iucC, so far unidentified, performs the second and final step in this reaction. This is based on the chemical characterization of two precursor hydroxamic acids (N epsilon-acetyl-N epsilon-hydroxylysine and N alpha-citryl-N epsilon-acetyl-N epsilon-hydroxylysine) isolated from a strain carrying a 0.3-kilobase-pair deletion in the iucC gene. The results support the existence of a biosynthetic pathway in which aerobactin arises by oxygenation of lysine, acetylation of the N epsilon-hydroxy function, and condensation of 2 mol of the resulting aminohydroxamic acid with citric acid.     

Temperature and heat production patterns inside organism clusters

Clustering of organisms under cold air temperature conditions is modelled with a finite-difference method. Metabolic functions of temperature are used to simulate completely ectothermic, completely endothermic, and other organisms. To adequately match real conditions, the core temperature is kept constant at a high level, while the periphery of the organism cluster is assigned a lower temperature representing the cold conditions under which clustering is observed for organisms. The numerical model reasonably predicts the observed temperature distribution in honeybee clusters. The results do not support suggestions that organisms could overheat in the core of a cluster if they do not use thermoregulatory mechanisms to cool down. Endothermic organisms are not as efficient as ectothermic ones in heating a cluster core temperature to a given level. The general ectothermic metabolic rate function exhibited one of the highest efficiencies for heating the cluster.     

The modified method for isolation and culture of highly homogeneous Leydig cell population from rat testes

A modified method for isolation and culture of a pure population of rat Leydig cells is described. For obtaining crude interstitial cell suspension, decapsulated testes were dispersed in 0.02% collagenase solution in Ca2+, Mg2+--free Hanks medium for 1 hour. Then, approx. 5 X 10(7) cells were centrifuged in 10-90% discontinuous, isoosmotic Percoll gradient at 3000 g for 20 min. The cells from eight fractions obtained were collected and cultured in Eagle's MEM for 4 days. Using morphological methods, 1.059-1.070 g/ml density fraction contained 97% and 1.070-1.080 g/ml fraction contained 90% viable Leydig cells. The cells secreted testosterone to the culture medium and responded to LH stimulation with over four-fold increase in hormone secretion.     

Components and Controls of Water Flux in an Old-growth Douglas-fir–Western Hemlock Ecosystem

We report measurements of rates of sap flow in dominant trees, changes in soil moisture, and evaporation from coarse woody debris in an old-growth Douglas-fir–western hemlock ecosystem at Wind River, Washington, USA, during dry periods in summer. The measurements are compared with eddy-covariance measurements of water-vapor fluxes above the forest (E_e) and at the forest floor (E_u) to examine the components of ecosystem water loss and the factors controlling them. Daily values of E_u were about 10% of E_e. Evaporation from coarse woody debris was only about 2% of E_e. Transpiration (E_t), estimated by scaling sap-flow measurements accounted for about 70% of (E_e– E_u); transpiration from subdominant trees may account for the remainder. The daily total change in soil moisture (E_s) in the top 30 cm was larger than the net change, probably because of hydraulic redistribution of soil water by roots. Observed differences between E_s and E_e were probably because roots also extract water from greater depth, and/or because the measuring systems sample at different spatial scales. The ratio of E_t to E_s decreased with decreasing soil water content, suggesting that partitioning in water use between understory and overstory changed during the season. The rate of soil drying exceeded E_e early in the day, probably because water vapor was being stored in canopy air space and condensed or adsorbed on tree stems, lichens, and mosses. The daily variation of E_e with vapor-pressure deficit showed strong hysteresis, most likely associated with transpiration of water stored in tree stems and branches.