ROS production and antioxidative system activity in embryonic axes of <Emphasis Type="Italic">Quercus robur</Emphasis> seeds under different desiccation rate conditions

The seeds of pedunculate oak (Quercus robur L.) were subjected to slow (S) and rapid (R) desiccation at desiccation rates of 0.16 and 0.39% H₂O per hour, respectively. Till ca. 40% water content (WC) the germination capacity of seeds in the S and R variants was high (ca. 100%). Between 40 and 28% WC, germination capacity declined to 20 and 50% in S and R variants, respectively. The decrease in seed viability was accompanied by a significant increase of electrolyte leakage from embryonic axes (28% for S and 15% for R variants). In the embryonic axes of seeds subjected to slow desiccation, malondialdehyde (MDA) and free fatty acid (FFA) contents were significantly higher than those in R variants, indicating greater membrane damage due to lipid peroxidation. The production of ROS (H₂O₂ and O₂^·−) was significantly higher in S than in R variants. The low molecular weight antioxidants α-tocopherol, ascorbic acid (ASA), and phenolic compounds indicated different reactions in response to desiccation stress. ASA levels decreased during desiccation to a similar degree in both the S and R variants. A significant decrease of total phenols was observed in R variant, which coincided with a significant increase of guaiacol peroxidase (POX) activity. α-Tocopherol content was significantly higher in the embryonic axes of seeds subjected to rapid drying. The activities of the enzymatic scavengers APX and GR had similar runs and were slightly higher in R variant. The activities of POX and SOD were significantly higher in the embryonic axes of seeds subjected to rapid drying. These results show that rapid dehydration of Q. robur seeds leads to the greater mobilization of antioxidant system in embryonic axes, particularly increased levels of α-tocopherol and POX and SOD activities, in the first stages of water loss. This mobilization has a greater impact on maintenance of higher viability of seeds after drying to lower level of WC.     

Insights into the smooth‐to‐rough transitioning in Mycobacterium bolletii unravels a functional Tyr residue conserved in all mycobacterial MmpL family members

In mycobacteria, MmpL proteins represent key components that participate in the biosynthesis of the complex cell envelope. Whole genome analysis of a spontaneous rough morphotype variant of Mycobacterium abscessus subsp. bolletii identified a conserved tyrosine that is crucial for the function of MmpL family proteins. Isogenic smooth (S) and rough (R) variants differed by a single mutation linked to a Y842H substitution in MmpL4a. This mutation caused a deficiency in glycopeptidolipid production/transport in the R variant and a gain in the capacity to produce cords in vitro. In zebrafish, increased virulence of the M. bolletii R variant over the parental S strain was found, involving massive production of serpentine cords, abscess formation and rapid larval death. Importantly, this finding allowed us to demonstrate an essential role of Tyr842 in several different MmpL proteins, including Mycobacterium tuberculosis MmpL3. Structural homology models of MmpL4a and MmpL3 identified two additional critical residues located in the transmembrane regions TM10 and TM4 that are facing each other. We propose that these central residues are part of the proton‐motive force that supplies the energy for substrate transport. Hence, we provide important insights into mechanistic/structural aspects of MmpL proteins as lipid transporters and virulence determinants in mycobacteria.     

Dynamics of the Growth and Population Composition of Mixed Cultures of R,S, and M Dissociants of Pseudomonas aeruginosa

The population composition of polycultures of Pseudomonas aeruginosa dissociants (R + M and R + S + M) developing on media with various contents and ratios of nitrogen and phosphorus has been studied. Irrespective of its proportion (10 to 90%) in the inoculum, the R variant accounted for 65 to 84% of the whole population of linear-phase and stationary-phase binary cultures of R and M dissociants, which differ in terms of energy metabolism and nutritional requirements. After prolonged cultivation, the population in the binary culture contained only R cells (100%), which are characterized by minimum requirements with respect to the main biogenic elements. These data agree with the predictive data of model studies and can be attributed to regulation of the population composition of bacterial cultures by trophic factors. It was established that the proportion of M cells, which are distinguished by maximum nutrient requirements and enhanced stability, increased during two developmental stages of the Ps. aeruginosa polycultures (R + M and R + S + M): the lag phase and the decay stage. This result cannot be due to the influence of trophic factors and presumably results from changes in the levels of autoregulatory factors (anabiosis autoinducers) involved in stress resistance and plausibly in the adaptive interconversion of dissociants upon transfer to a new medium (during the lag phase) and under starvation conditions (at the onset of the decay phase).__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 475–482.Original Russian Text Copyright © 2005 by Mil’ko, Khabibullin, Nikolaev, Kozlova, El’-Registan.     

The Role of Melanocortin‐1 Receptor Polymorphism in Skin Cancer Risk Phenotypes

We have examined melanocortin‐1 receptor (MC1R) variant allele frequencies in the general population and in a collection of adolescent dizygotic and monozygotic twins to determine statistical associations of pigmentation phenotypes with increased skin cancer risk. This included hair and skin color, freckling, mole count and sun exposed skin reflectance. Nine variants were studied and designated as either strong R (OR = 63; 95% CI 32–140) or weak r (OR = 5; 95% CI 3–11) red hair alleles. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. To assess the interaction of the brown eye color gene BEY2/OCA2 on the phenotypic effects of variant MC1R alleles we imputed OCA2 genotype in the twin collection. A modifying effect of OCA2 on MC1R variant alleles was seen on constitutive skin color, freckling and mole count. In order to study the individual effects of these variants on pigmentation phenotype we have established a series of human primary melanocyte strains genotyped for the MC1R receptor. These include strains which are MC1R wild‐type consensus, variant heterozygotes, and homozygotes for strong R alleles Arg151Cys and Arg160Trp. Ultrastructural analysis demonstrated that only consensus strains contained stage III and IV melanosomes in their terminal dendrites whereas Arg151Cys and Arg160Trp homozygous strains contained only immature stage I and II melanosomes. Such genetic association studies combined with the functional analysis of MC1R variant alleles in melanocytic cells should provide a link in understanding the association between pigmentary phototypes and skin cancer risk.     

Directed Evolution of E. coli Alkaline Phosphatase Towards Higher Catalytic Activity

Directed evolution was used to enhance the catalytic activity of E. coli alkaline phosphatase (EAP). Through two rounds of error-prone PCR and one round of DNA shuffling followed by a rapid, sensitive screening procedure, several improved variants were obtained. Their enzymatic kinetic properties, thermal stabilities and possible mechanism for the improvement were investigated. In 1.0 M Tris buffer, the specific activity of the most active EAP variant S2163 was 1500 units/mg protein, showing it to be 3.6 times more active than the D101S parent enzyme and ～40 times more active than the wild-type EAP. At the same time, the K_m value of the S2163 variant decreased to 1491 μM from the 2384 μM of the D101S. As a result, the k_cat/K_m ratio of this variant showed a 5.8-fold enhancement over that of D101S parent enzyme. Three activating amino acid substitutions, K167R, G180S and S374C, which were located far away from the center of the catalytic pocket, were identified by sequencing the genes encoding evolved enzymes. Possible explanations for the improvement of activity were analyzed.     

Missense mutations and polymorphisms of the <Emphasis Type="Italic">MC4R</Emphasis> gene in Polish obese children and adolescents in relation to the relative body mass index

Extensive studies of the MC4R gene polymorphism showed that, among numerous variants, there are mutations responsible for monogenic obesity, as well as polymorphisms negatively correlated with the risk of obesity. In this report, we present the first studies of the whole coding sequence of the MC4R gene in 243 Polish obese children and adolescents (the mean relative body mass index [RBMI] was 163.6). In addition, 101 non-obese adults were also analyzed. Direct sequencing facilitated the identification of six missense (K73R, V103I, T112M, S127L, M215L, and I251L) and one silent (c.756 C > T) single-nucleotide polymorphisms (SNPs). Two non-synonymous polymorphisms (K73R and M215L) appeared to be novel and one was found in obese patients (M215L, one patient) and one in non-obese adults (K73R, one person). The overall frequency of non-synonymous variant carriers reached 4.1% and 6.9% in obese patients and non-obese adults, respectively. Only one obesity-associated variant (127L) was found in two obese patients (0.82%) and in two non-obese adults (1.98%). The obesity-protecting variants (103I and 251L) appeared to be the most common in both groups: 3.3% and 4.0%, respectively. It was also observed that the RBMI in obese children and adolescents carrying the minor variants did not differ significantly from the non-carriers; however, the expected trends for the associated and protecting variants were observed. We conclude that the contribution of the MC4R gene variants to the pathogenesis of obesity in Polish children and adolescents is low.     

Engineering of the yeast antioxidant enzyme Mpr1 for enhanced activity and stability

The budding yeast Saccharomyces cerevisiae Σ1278b has the MPR1 gene, which confers resistance to the proline analogue azetidine‐2‐carboxylate (AZC). This gene encodes an N‐acetyltransferase Mpr1 that detoxifies AZC, and the homologous genes have been found in many yeasts. Recently, we found that Mpr1 protects yeast cells by reducing the intracellular reactive oxygen species (ROS) levels under oxidative stresses, such as heat‐shock, freezing, or ethanol treatment. Unlike the known antioxidant enzymes, Mpr1 is thought to acetylate toxic metabolite(s) involved in ROS generation via oxidative events. To improve the enzymatic functions of Mpr1, we applied PCR random mutagenesis to MPR1. The mutagenized plasmid library was introduced into the S. cerevisiae S288C strain lacking MPR1, and we successfully isolated two Mpr1 variants with higher AZC resistance (K63R and F65L/L117V). Interestingly, overexpression of the K63R variant was found to increase cell viability or decrease intracellular ROS levels after exposure to H₂O₂ or ethanol compared with the wild‐type Mpr1. In vitro studies with the recombinant enzymes showed that the catalytic efficiency of the K63R variant for AZC and acetyl‐CoA was higher than that of the wild‐type Mpr1 and that the F65L mutation greatly enhanced the thermal stability. The mutational analysis and molecular modeling suggest that an α‐helix containing Lys63 and Phe65 has important roles in the function of Mpr1. In addition, the wild‐type and K63R variant Mpr1 reduced intracellular ROS levels under ethanol stress conditions on haploid sake yeast cells. These results suggest that engineering Mpr1 might be useful in breeding oxidative stress‐tolerant yeast strains. Biotechnol. Bioeng. 2009;103: 341–352. © 2009 Wiley Periodicals, Inc.     

Effects of common atopy-associated amino acid substitutions in the IL-4 receptor alpha chain on IL-4 induced phenotypes

The human IL-4 receptor alpha chain gene (IL4R) is highly polymorphic and controversial reports have been published with respect to the association of different single nucleotide polymorphisms (SNPs) with atopy markers. Here we analyzed the functional and associational relevance of common IL4R coding SNPs. Transfection of B cell lines expressing the IL-4R variant V75+R576 did not result in enhanced IL-4 induced CD23 expression compared to cell lines expressing the wild type IL-4R alpha chain. Transfection of the IL-4R variant P503 into a murine T cell line did not influence IL-4 induced T-cell proliferation compared to wild type constructs. Analysis of six IL4R coding SNPs (I75V, E400A, C431R, S436L, S503P, Q576R) and common haplotypes (frequency 0.05%) in blood donors (n=300) did not indicate a significant association with elevated serum IgE level. Moreover, the most informative IL4R coding SNPs (I75V, C431R, Q576R) and related two- and three-point haplotypes (frequency 0.05%) were analyzed in a second, extended group of blood donors (n=689). Again, no significant association with elevated serum IgE was detectable. We conclude that common coding SNPs in the IL4R gene are unlikely to contribute significantly to increased IgE levels and variations outside the coding region may influence atopy susceptibility.     

LRRK2 R1628P increases risk of Parkinson’s disease: replication evidence

We showed that the frequency of a LRRK2 variant (c.4883G > C, R1628P) was higher in Parkinson’s disease (PD) compared to controls (8.4 vs. 3.4%, P = 0.046, OR 2.5, 95% CI 1.1–5.6). In the multivariate logistic regression (with adjustments made for the effect of age, age of onset, and gender), the heterozygous R1628P genotype was associated with an increased risk of PD compared to controls (OR 3.3, 95% CI 1.4– 7.9, P = 0.007). We provided an independent confirmation that the R1628P variant increases the risk of PD among Chinese.     

Requirements for Glucose, Nitrate, and Phosphate and Their Variations in the Analysis of the Mixed Culture of Pseudomonas aeruginosa Variants

The calibration data and sensitivity analysis for the variational model of Pseudomonas aeruginosa mixed culture containing R, S, and M phenotypic variants are presented. The model with calibrated parameters (requirements of the variants for glucose, nitrates, and phosphates) adequately describes the experiment. The sensitivity indices (the species abundances and total community size) changed least under the influence of the parameter variations in the one-factor limitation areas. The variant proportions are more sensitive to variation of the requirements than the community size.     

Test of agronomic characteristics and amplified fragment length polymorphism analysis of new rice germplasm developed from transformation of genomic DNA of distant relatives

New rice lines, restorer line RB207 and maintainer line Yewei B, with better agronomic traits were separately developed from variant progeny of R207 (rice restorer line) and V20B (rice maintainer line) through transformation of genomic DNA ofEchinochloa crusgalli (C₄ plant) andOryza minuta, respectively. The phenotypes of the variant lines were apparently different from those of the receptors. Yewei B had stronger tolerance to high temperature than did V20B. The number of spikelets per panicle and the 1000-grain weight of RB207 increased by 40% over those of R207. The results of amplified fragment length polymorphism (AFLP) analysis indicated that the polymorphism rates were both 4.4% between genomes of the variant lines and their receptors. Results demonstrated that special DNA segments fromE. crusgalli andO. minuta might integrate into the genome of cultivated rice and could be stably passed on. The study further shows that transformation of genomic DNA of distant relatives is an effective approach for creating new rice germ plasm. These authors contributed equally to this work.     

降水变化对红砂-珍珠碳、氮、磷化学计量特征的影响

以荒漠C3植物红砂(Reaumuria soongarica)和C4植物珍珠(Salsola passerina)为材料,在西北干旱荒漠区沿自然降水梯度,对不同降水条件下单生和混生红砂与珍珠根、茎、叶器官碳、氮、磷化学计量指标进行测定,分析其在不同生境下化学计量特征对种间关系及环境胁迫的响应规律。结果表明:(1)随干旱胁迫程度增加(降水量的减少),红砂各器官C含量平均升高7.73%,N、P含量分别平均降低6.20%、10.61%;珍珠各器官C含量平均升高7.36%,N、P含量分别平均降低5.93%、14.03%。两种植物叶片C含量升高表明其光合速率较低,生长缓慢,但对外界不利环境的防御能力增强,能更好地适应干旱环境。(2)干旱胁迫改变了红砂和珍珠的N、P含量在各器官的分配模式,两种植物N、P含量在叶部高于根部,在根、叶中N/P明显高于茎,表明两种植物不同器官受到的养分限制不同。(3)红砂各器官C、N、P含量高于珍珠,说明红砂防御能力较强,生长速率高,对资源的竞争和利用能力较珍珠强;珍珠C/N和C/P均高于红砂,表明珍珠比红砂有较强的碳同化能力和较高的营养利用效率。(4)在干旱胁迫条件下,红砂和珍珠均表现为碳素积累、氮磷素限制的格局,它们对于氮和磷的养分利用不活跃,受到氮和磷养分的限制较为均衡。     

Spontaneous duplication of a 661 bp element within a two-component sensor regulator gene causes phenotypic switching in colonies of Pseudomonas tolaasii, cause of brown blotch disease of mushrooms

Spontaneous sectoring of Pseudomonas tolaasii colonies results in a phenotypic switch from the smooth, pathogenic form (designated 1116S) to the rough non-pathogenic form (designated 1116R). This phenotypic switch can also be induced by mutation of the pheN master regulatory locus, which encodes a 99 kDa protein with homology to the conserved family of sensor regulator proteins. Southern blot analysis of genomic DNA from 1116S and 1116R probed with a 3.4 kb XhoI–BamHI fragment containing the pheN gene has revealed restriction fragment length polymorphisms in the pheN locus of 1116R. In order to characterize the genetic basis of this variation, the pheN locus (designated pheN′ ) was cloned from 1116R and its nucleotide sequence determined. A 661 bp duplication was identified within pheN′ introducing a frameshift mutation in the predicted pheN open reading frame (ORF). A resulting predicted ORF of pheN′ designated ORF2 encodes a polypeptide of 706 amino acid residues, with a predicted molecular weight of 77 kDa, and which lacks part of the PheN sensor domain. Southern blot analysis of genomic DNA using a probe within the duplicated sequence revealed the presence of two bands in 1116R but only one band in the 1116S form. Polymerase chain reaction (PCR) analysis of 25 independently isolated 1116R sectors using primers flanking the duplication site in pheN confirmed the presence of the duplicated 661 bp sequence within this region in all of the sectors and the absence of the duplicated sequence in spontaneous revertants from 1116R to 1116S. Northern blot analysis of RNA from 1116S and 1116R using a pheN probe showed that ORF2 was transcribed in the 1116R form. The presence of a truncated PheN protein in 1116R was verified by Western blot analysis of total cell protein using a LemA antiserum, which revealed the presence of 99 kDa and 77 kDa cross-reactive bands in 1116S and 1116R respectively. It is concluded that the spontaneous colony-sectoring event that results in the 1116R phenotypic variant form of P. tolaasii arises owing to a 661 bp DNA duplication within the 5′ end of the pheN gene, which results in loss of the periplasmic sensor domain of PheN and elimination of normal PheN function.     

Genetic polymorphisms and haplotypes of the organic cation transporter 1 gene (SLC22A1) in the Xhosa population of South Africa

Human organic cation transporter 1 is primarily expressed in hepatocytes and mediates the electrogenic transport of various endogenous and exogenous compounds, including clinically important drugs. Genetic polymorphisms in the gene coding for human organic cation transporter 1, SLC22A1, are increasingly being recognized as a possible mechanism explaining the variable response to clinical drugs, which are substrates for this transporter. The genotypic and allelic distributions of 19 nonsynonymous and one intronic SLC22A1 single nucleotide polymorphisms were determined in 148 healthy Xhosa participants from South Africa, using a SNAPshot^® multiplex assay. In addition, haplotype structure for SLC22A1 was inferred from the genotypic data. The minor allele frequencies for S14F (rs34447885), P341L (rs2282143), V519F (rs78899680), and the intronic variant rs622342 were 1.7%, 8.4%, 3.0%, and 21.6%, respectively. None of the participants carried the variant allele for R61C (rs12208357), C88R (rs55918055), S189L (rs34104736), G220V (rs36103319), P283L (rs4646277), R287G (rs4646278), G401S (rs34130495), M440I (rs35956182), or G465R (rs34059508). In addition, no variant alleles were observed for A306T (COSM164365), A413V (rs144322387), M420V (rs142448543), I421F (rs139512541), C436F (rs139512541), V501E (rs143175763), or I542V (rs137928512) in the population. Eight haplotypes were inferred from the genotypic data. This study reports important genetic data that could be useful for future pharmacogenetic studies of drug transporters in the indigenous Sub-Saharan African populations.     

Identification of novel functional variants of the melanocortin 1 receptor gene originated from Asians

Human melanocortin 1 receptor (MC1R) is a seven transmembrane G-coupled protein receptor that upregulates the cAMP pathway. Several functional variants of MC1R that show an impaired ability to activate the cAMP pathway are strongly associated with fair skin and red hair in Europeans and European descendants. The sequence variations of the MC1R gene were repeatedly investigated against worldwide populations; however, there was no evidence that functional variant of MC1R exists in non-European descendants. We report the presence of novel functional variants of MC1R with Asian origins. Three novel variants of MC1R, Phe147Δ, Thr157Ile, and Pro159Thr, were identified in our screening for the sequence variations of the MC1R gene against 995 individuals from 30 Asian and Oceanian populations; there was a single case for the Pro159Thr variant allele and two instances of Phe147Δ and Thr157Ile variant alleles. Our pharmacological assay revealed that Phe147Δ, Thr157Ile, and Pro159Thr variant showed similar or more dramatically impaired activities in comparison with Arg151Cys, which is a major functional variant of MC1R in Europeans. These functional variant alleles were geographically localized in relatively high latitudes, which suggest that the adaptation to ambient UV light intensity may play an important role in shaping the geographical distribution of MC1R alleles in Asia and Oceania.     

Metabolism of amino acids in germinating yellow lupin seeds III. Breakdown of arginine in sugar-starved organs cultivated in vitro

The pathways of arginine transformations in organs of yellow lupin (Lupinus luteus L.) cultivated in vitro in the presence and absence of sucrose were investigated. Isolated embryo axes, isolated cotyledons and seeds deprived of their coat were cultured for 96 h on Heller medium with 60 mM saccharose (the fed variant, +S), without sugar (the starved variant, −S) and for 72 h without sugar, followed by 24 h in its presence (the transferred variant, −S→+S). Activities of arginine decarboxylase [EC 4.1.1.19], arginase [EC 3.5.3.1], and urease [EC 3.5.1.5] were assessed in extracts from isolated embryo axes. They were the highest in the sugar-starved variant. Supplementation of the medium with saccharose resulted in decrease in enzyme activities. The level of urea was higher (of ca. 20 %) in starved embryos than in embryos grown in the saccharose-containing medium. Moreover, participation of transamination in arginine catabolism was evidenced.     

Role of gramicidin S in the sporulation of the R+ and R- variants of Bacillus brevis var. G.-B.]

The effect of gramicidin S added to the cultivation medium on sporulation of the gramicidin S-producing P+ variant and gramicidin S-nonproducing P- variant of Bacillus brevis var. G.-B. was studied. Gramicidin S added to the synthetic medium with glucose in an amount of 30 and 100 microgram/ml 4 and 7 hours after inoculation with the vegetative cells of R- variant had no effect on the growth of the culture but retarded its sporulation. When gramicidin S was added in an amount of 100 microgram/ml 4 hours after inoculation, the sporulation rate of R- variant strongly decreased, rohile sporulation was not suppressed as it was noted before with respect to R+ variant. Active stimulation of Bacillus brevis var. G.-B. sporulation was observed after addition of gramicidin S 13 hours after development of R+ and R- variants without the antibiotic biosynthesis. Synthesis of gramicidin S by R+ strain was suppressed by the specific inhibitor beta-phenyl-beta-alanine. The amount of gramicidin S added to the medium during the sporulation process of R+ and R- variants decreased. On addition of 30 microgram/ml of the antibiotic it was practically not detectable when the culture showed the greatest number of the spores. Therefore, gramicidin S added to the medium is probably adsorbed by the cells of Bac. brevis var. G.-B. and affects sporulation of R- and R+ variants thus accelerating or retarding this process depending on the cultivation conditions.     

Misfolding, degradation, and aggregation of variant proteins. The molecular pathogenesis of short chain acyl-CoA dehydrogenase (SCAD) deficiency

Short chain acyl-CoA dehydrogenase (SCAD) deficiency is an inborn error of the mitochondrial fatty acid metabolism caused by rare variations as well as common susceptibility variations in the SCAD gene. Earlier studies have shown that a common variant SCAD protein (R147W) was impaired in folding, and preliminary experiments suggested that the variant protein displayed prolonged association with chaperonins and delayed formation of active enzyme. Accordingly, the molecular pathogenesis of SCAD deficiency may rely on intramitochondrial protein quality control mechanisms, including degradation and aggregation of variant SCAD proteins. In this study we investigated the processing of a set of disease-causing variant SCAD proteins (R22W, G68C, W153R, R359C, and Q341H) and two common variant proteins (R147W and G185S) that lead to reduced SCAD activity. All SCAD proteins, including the wild type, associate with mitochondrial hsp60 chaperonins; however, the variant SCAD proteins remained associated with hsp60 for prolonged periods of time. Biogenesis experiments at two temperatures revealed that some of the variant proteins (R22W, G68C, W153R, and R359C) caused severe misfolding, whereas others (R147W, G185S, and Q341H) exhibited a less severe temperature-sensitive folding defect. Based on the magnitude of in vitro defects, these SCAD proteins are characterized as folding-defective variants and mild folding variants, respectively. Pulse-chase experiments demonstrated that the variant SCAD proteins either triggered proteolytic degradation by mitochondrial proteases or, especially at elevated temperature, aggregation of non-native conformers. The latter finding may indicate that accumulation of aggregated SCAD proteins may play a role in the pathogenesis of SCAD deficiency.     

Phenotypic variability in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains Sp7 and Sp245: Association with dormancy and characteristics of the variants

The state of metabolic dormancy in diazotrophic bacteria Azospirillum brasilense Sp7 (non-endophytic strain) and Sp245 (endophytic strain) was found to be associated with phenotypic variability. The latter manifested itself in the extension of the spectrum of A. brasilense phenotypic variants resulting from plating of cyst-like resting cells (CRC) on solid media and was more pronounced in strain Sp7. The major colony’s morphological variants of strain Sp7 were (1) the dominant S type; (2) the highly pigmented Pg type; (3) the R type; (4) the Sm type, forming small colonies; and (5) the Sg type, forming segmented colonies. In addition to their colony morphology, the variants differed in the phenotype stability during transfers on the standard solid medium and in their motility in semisolid agar. The occurrence frequency of the phenotypic variants depended on the conditions and duration of incubation (storage) of the CRC of strain Sp7, as well as on heat treatment (at 55 and 60°C for 10 min) of the cells prior to inoculation. The maximum frequency of S → Pg transitions (up to 74%) was observed during the germination of CRC stored in a spent culture medium at −20°C for 4 months; the maximum frequency (up to 100%) of S → Sm transitions was observed after inoculation of the CRC subjected to heat treatment. The Pg variants were the most stable, whereas other types reverted rapidly to the S or Pg variant. The S variant grown in semisolid agar exhibited the mixed type of motility (Swa⁺Gri⁺, swarming and migration in the form of microcolonies); the Pg and Sg variants showed the Swa⁺Gri⁻ (swarming) phenotype and the Sm variant was nonmotile (Swa⁻Gri⁻ phenotype). The spectrum of phenotypic variants of the endophytic strain Sp245 was narrower than that of strain Sp7 and was represented by S, Sm, and M (mucoid) variants that differed in the patterns of cell motility: the dominant S type displayed the swarming pattern (Swa⁺Gri⁻), the mucoid M type showed the mixed type (Swa⁺Gri⁺) of motility, and the Sm variant was nonmotile. The differences between the nonendophytic strain Sp7 and the endophytic strain Sp245 in their capacity for phenotypic dissociation and cell motility in semisolid media may reflect their ability to adapt to changing ambient conditions and specificity of plant-microbial interactions.     

Evolutionary History of 4.5SI RNA and Indication That It Is Functional

To date, the small nuclear 4.5S_I RNA has only been studied in the rat (Rattus norvegicus). Combining PCR and hybridization analyses, we have revealed 4.5S_I RNA homologues sequences in the genomes of four myomorph rodent families (Muridae, Cricetidae, Spalicidae, and Rhizomyidae), and not in other myomorph families (Dipodidae, Zapodidae, Geomyidae, and Heteromyidae) or sciuromorph and caviomorph rodents. By Northern-hybridization, 4.5S_I RNA has been detected in the common rat (R. norvegicus, Muridae), golden hamster (Mesocricetus auratus, Cricetidae), and Russian mole rat (Spalax microphthalmus, Spalacidae), but not in the related great jerboa (Allactaga jaculus, Dipodidae) or in four non-myomorph rodent species tested. cDNA derived from 4.5S_I RNA of M. auratus and S. microphthalmus has been cloned and sequenced. The hamster RNA is found to differ from rat 4.5S_I RNA by only one nucleotide substitution. For the mole rat, two variants of 4.5S_I RNA are detected: short (S) and long (L) with length 101 and 108 nt, respectively. The L variant differs from the S variant as well as from murid and cricetid 4.5S_I RNAs by both a 7 nt insertion and a varying number of nucleotide substitutions. The sequence similarity between the spalacid S-variant and murid/crecitid variants of 4.5S_I RNA is 90%. Judging from species distribution, 4.5S_I RNA genes emerged during the same period of time as the related short interspersed element B2 arose. This occurred after the divergence of Dipodidae lineage but before the branching of Spalicidae/Rhizomyidae lineage from a common myomorph rodent stem. S variant genes seemed to emerge in a common ancestor of spalacids and rhizomyds whereas L variant genes formed in spalacids following the divergence of these two families. The low rate of evolutionary changes of 4.5S_I RNA, at least, in murids and cricetids (6 × 10⁻⁴ substitutions per site per million years), suggests that this RNA is under selection constraint and have a function. This is a remarkable fact if the recent origin and narrow species distribution range of 4.5S_I RNA genes is taken into account. Genes with narrow species distribution are proposed to be referred to as stenogenes. Received: 11 December 2000 / Accepted: 27 August 2001