Why is the polymerase chain reaction resistant to in vitro evolution?

A variety of methods have been developed to amplify DNA and RNA. These methods vary in their susceptibility to evolve new molecular species differing from the starting template. PCR is exceptionally resistant to in vitro evolution, whereas methods such as Q replicase and 3SR are much less robust. This paper develops some simple mathematical models which suggest that PCR is resistant to in vitro evolution because the reaction controls replication in discrete cycles: fast replication is of little advantage during PCR because the reaction limits fast replicators as well as slow ones to a single copy per cycle. In contrast, continuous (isothermal) reactions, as in the Q replicase reaction, favor fast replicators. The advantage of fast replication is compounded in continuous reactions, because a fast replicator can complete many generations of replication during the time it takes a slow replicator to complete one generation. These models suggest that continuous amplication protocols will never achieve the robustness against in vitro evolution observed with PCR.Correspondence to: J.J. Bull     

Comparisons of the Genomes of New Giant Phages Isolated from Environmental Pseudomonas aeruginosa Strains of Different Regions

To study the genome diversity of bacteriophages from geographically distant natural populations, new giant KZ-like Pseudomonas aeruginosa phages isolated in two different regions were compared with earlier known phages of three species (KZ, Lin68, EL). A broad spectrum of lytic activity was demonstrated for all KZ-like phages. Phages of the KZ species proved to be common in natural populations of various regions, while EL- and Lin68-related phages were extremely rare. Most KZ-related phages had unique DNA restriction patterns, but the differences between these were only minor, and the genomes did not contain nonhomologous fragments. The spectrum of capsid polypeptides proved to be conserved in each species, and was proposed as a character necessary and sufficient for express classification of phages with an accuracy of species. Phages isolated in different geographical regions showed no substantial difference. Some phages only slightly differing in DNA restriction pattern from KZ may be used to study the origin of KZ genes coding for orthologs of proteins of unrelated species (other phages, pathogenic bacteria, eukaryotes).     

Anti-diabetic activity of <Emphasis Type="Italic">β</Emphasis>-glucans and their enzymatically hydrolyzed oligosaccharides from <Emphasis Type="Italic">Agaricus blazei</Emphasis>

-Glucans were prepared from Agaricus blazei Murill by repeated extraction with hot water. The average molecular weights of -glucans were 30–50 kDa by gel filtration chromatography. Oligosaccharides (AO), derived from hydrolyzing -glucans with an endo--(16)-glucanase from Bacillus megaterium, were mainly di- and tri-saccharides. Though -glucans and AO both showed anti-hyperglycemic, anti-hypertriglyceridemic, anti-hypercholesterolemic, and anti-arteriosclerotic activity indicating overall anti-diabetic activity in diabetic rats, AO had about twice the activity of -glucans with respect to anti-diabetic activity.     

Polymorphism of the HLA DR1 haplotype in the Israeli population investigated at the serological,cellular, and genomic levels

In the present report, we used serological, cellular, and restriction fragment length polymorphism (RFLP) to investigate the DR1 haplotype in the Israeli population. We describe an Israeli homozygous typing cell (HTC), HLA-DwLVA, which defines a new lymphocyte-activating determinant associated with Bw65, DR1 and distinct from Dwl. The parents of this donor, non-Ashkenazi Algerian Jews, are first cousins and share HLA-Cw8, Bw65, BfS, DR1, DQw1, DPw4. No specificity could be assigned to HLA-DwLVA using the 91 Ninth Workshop HTCs. Two families and forty unrelated DR1 individuals were studied with DwLVA and a panel of DR1/Dw1 HTCs. HLA-DwLVA showed segregation as a single determinant within families. This new specificity was present in 24 out of 40 (60%) unrelated DR1 individuals, indicating that in the Israeli population DwLVA is the main lymphocyte-defined determinant associated with the serologically defined DRI specificity, in contrast to non-Jewish Caucasoids where DR1 is significantly associated with Dw1. The vast majority of DwLVA-positive carriers were also Bw65 carriers, indicating that Bw65, DR1, DwLVA may represent a typical allele combination in the Israeli population. The RFLP analysis established the correlation of certain RFLPs with Dw1 and DwLVA. In addition, we describe a cluster of RFLPs that may correspond to a new Dw subtype associated with DR1, for which no serological and cellular reagents have been described so far.     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo     

A method of line development using doubled haploids: the single doubled haploid descent recurrent selection

Summary Different methods of line development using doubled haploids in recurrent selection are presented. They are divided into two types: recurrent selection with progeny testing and recurrent selection on the phenotype of lines. It is shown that one of the best methods of line development is based on single doubled haploid descent recurrent selection. Only one line is studied per plant of the population, and the best lines are intercrossed. Using this method it is very easy to extract lines for variety development.     

Induction of the Synthesis of Endo-1,4-β-xylanase and β-Galactosidase in Original and Recombinant Strains of the Fungus Penicillium canescens

The induction of synthesis of the secreted enzymes endo-1,4--xylanase (EC 3.2.1.8) and -galactosidase (EC 3.2.1.23) in original and recombinant Penicillium canescens strains has been studied. In all producer strains, the synthesis of these enzymes was induced by arabinose and its metabolite arabitol. The two enzymes differed in the concentration of arabinose required for induction: the synthesis of -galactosidase was most pronounced at 1 mM, whereas maximum synthesis of endo-1,4--xylanase was observed at 5–10 mM. An increase in the number of endo-1,4--xylanase copies in the high-copy-number strain of the fungus suppressed the synthesis of -galactosidase; the synthesis of endo-1,4--xylanase in the high-copy-number recombinant producing -galactosidase was affected to a lesser extent. The amount of enzymes synthesized did not depend on the saccharide used as the sole source of carbon for growing the mycelium prior to its transfer to the inducer-containing medium.     

In vitro comparison of ceftazidime,aztreonam, cefpirome,gentamicin, imipenem,enoxacin, and ticarcillin plus clavulanic acid against 108 strains ofPseudomonas maltophilia

Pseudomonas maltophilia is an uncommon cause of hospital-acquired infection and is resistant to most of the antimicrobial agents used in the treatment of gram-negative infections. Susceptibility of 108 isolates ofP. maltophilia to ceftazidime, aztreonam, defpirome, gentamicin, imipenem, enoxacin, and ticarcillin plus clavulanic acid was determined by an agar dilution method. The isolates were in general resistant to the antibiotics. Imipenem and cefpirome were not active at clinically achievable levels. Of the isolates, 20% were susceptible to 16 g/ml ceftazidime, 53% were susceptible to 4 g/ml enoxacin, 10% were susceptible to 4 g/ml gentamicin, and 25% were susceptible to 64 g/ml ticarcillin plus 2 g/ml clavulanic acid.     

Larval competition in Drosophila melanogaster: frequency-dependence of viability

Summary The application of the overfeeding technique (interruption of the competition during larval development) to the study of larval competition in two-strain cultures of Drosophila melanogaster demonstrates the following points: (1) viability is a function of competition time; (2) viability becomes more frequency-dependent as competition time increases; (3) the dynamics of the inner subpopulation (adults that have passed all their development in a crowded condition) and outer subpopulation (adults coming from larvae recovered by interruption of competition) vary with time as regards frequency-dependence; and (4) the wild type strain Oregon is the active agent in competition with the strain cardinal.     

Axillary shoot induction and plant regeneration in Plantago ovata Forssk

Axillary shoot induction and plant regeneration were obtained in Plantago ovata. The optimum medium for inducing axillary shoots was Murashige & Skoog (MS) medium [5] supplemented with 4.6 M kinetin and 0.05 M NAA. Rooting of shoots was best on half-strength MS medium containing 5.0 M IBA and 0.05 M kinetin. The regenerated plants were similar to the control plants in karyotypic and phenotypic details.     

The origin of land plants: Phylogenetic relationships among charophytes,bryophytes, and vascular plants inferred from complete small-subunit ribosomal RNA gene sequences

Complete nuclear-encoded small-subunit 18S rRNA (=SSU rRNA) gene sequences were determined for the prasinophyte green alga Mantoniella squamata; the charophycean green algae Chara foetida, Coleochaete scutata, Klebsormidium flaccidum, and Mougeotia scalaris; the bryophytes Marchantia polymorpha, Fossombronia pusilla, and Funaria hygrometrica; and the lycopod Selaginella galleottii to get a better insight into the sequential evolution from green algae to land plants. The sequences were aligned with several previously published SSU rRNA sequences from chlorophytic and charophytic algae as well as from land plants to infer the evolutionary relationships for major evolutionary lineages within the Chlorobionta by distance matrix, maximum parsimony, and maximum likelihood analyses. Phylogenetic trees created by the different methods consistently placed the Charophyceae on the branch leading to the land plants. The Charophyceae were shown to be polyphyletic with the Charales (charalean algae) diverging earlier than the Coleochaetales, Klebsormidiales, Chlorokybales, and Zygnematales (charophycean algae) which branch from a point closer to the land plants in most analyses. Maximum parsimony and maximum likelihood analyses imply a successive evolution from charophycean algae, particularly Coleochaetales, to bryophytes, lycopods, and seed plants. In contrast, distance matrix methods group the bryophytes together with the charophycean algae, suggesting a separate evolution of these organisms compared with the club moss and the seed plants. Correspondence to.: V.A.R. Huss     

Adaptive Evolution of Scorpion Sodium Channel Toxins

Gene duplication followed by positive Darwinian selection is an important evolutionary event at the molecular level, by which a gene can gain new functions. Such an event might have occurred in the evolution of scorpion sodium channel toxin genes (- and -groups). To test this hypothesis, a robust statistical method from Yang and co-workers based on the estimation of the nonsynonymous-to-synonymous rate ratio ( = d_N/d_S) was performed. The results provide clear statistical evidence for adaptive molecular evolution of scorpion - and -toxin genes. A good match between the positively selected sites (evolutionary epitopes) and the putative bioactive surface (functional epitopes) indicates that these sites are most likely involved in functional recognition of sodium channels. Our results also shed light on the importance of the B-loop in the functional diversification of scorpion - and -toxins.     

Cloning of the dnaB gene of Escherichia coli: the dnaB gene of groPB534 and groPB612 and the replication of phage lambda

Summary Fragments of the E. coli chromosome that carry the dnaB groPB534 or groPB612 alleles have been cloned into a cosmid vector. The resulting recombinant plasmids contained the genes uvrA, groP (B534 or B612), and lexA. Further subcloning into high copy number plasmids, during which the uvrA and lexA genes were removed successively, yielded a groPB534 and groPB612 DNA fragment of about 2.4 kb each. Both fragments contained an overlapping 1.8 kb segment of DNA in which the sites of all restriction enzymes tested were identical. The size of these dnaB gene fragments were further delimited by deletion analysis.In E. coli groPB534 in which wild-type and A mutants do not replicate (Georgopoulos and Herskowitz 1971) phage replication is rescued if the strain contains the groPB534 gene on high copy number plasmids. On the contrary, in E. coli groPB612, which is temperature-sensitive for its groP character, replication of and A is abolished at 30° C if the strain contains the groPB612 recombinant plasmid. On the other hand, replication of B remains unaffected whether or not the groP strains harbor the isogenic dnaB gene-containing plasmid. The results suggest that within the cell not only the quality but also the relative amounts of dnaB and P protein are crucial for phage replication.     

Replication in Drosophila chromosomes

DNA fibre autoradiography of highly polytenized nuclei in salivary glands of Drosophila nasuta larvae reveals two distinct types of active replicons. Type I replicons are longer (mean size=64 m), have a very high rate of fork migration (average rate=0.95 m/min) and generally occur in large arrays often extending over several thousand m. In contrast, the type II replicons are smaller (mean size= 20 m), slow replicating (average rate=0.07 m/min) and occur in short arrays containing only a few closely spaced active replicons. Evidence is presented that type I replicons are active in the early S and type II in the late S. Observations on autoradiographic labelling of partially lysed polytene chromosomes provide evidence for a lack of temporal and spatial agreement in the activation of origin points in homologous regions of the lateral polytene strands; these observations also suggest local variations in levels of polyteny within a chromosome. On the basis of this and other available information on replication in polytene chromosomes the possible roles of the two replicon types in the generation of the different ³H-thymidine labelling patterns of polytene chromosomes are discussed.We take pleasure in dedicating this paper to our inspiring teacher Prof. S.P. Ray Chaudhuri on his completing 75 years of fruitful life     

Sucrose in storage media and cultivar affects post-storage regrowth of <Emphasis Type="Italic">in vitro</Emphasis> Hosta propagules

The goal of this research was to investigate if culturing in high sucrose (5%) liquid media during multiplication phase (stage II) would enhance endogenous sugar levels and dry matter sufficiently to allow storage of in vitro plants in sugar free media without adversely affecting post-storage recovery. Hosta tokudama Newberry Gold (NBG) and Hosta Striptease were cultured in Murashige and Skoog (MS) media containing 5% sucrose during stage II and transferred to rooting phase (stage III) in MS medium without (0%) sucrose or with 3% sucrose for 4weeks. At the end of stage III, cultures were stored, with the remaining media, at 10°C with 5molm^–2s^–1 photosynthetic photon flux (PPF) from cool white fluorescent lamps for 7 or 14weeks with or without a 2-week dark period prior to removal from storage. In both cultivars, stage III plants cultured in 3% sucrose media had higher soluble sugar levels and greater shoot and root biomass than those cultured in 0% sucrose media. Shoot and root soluble sugars decreased during storage. Shoot growth ceased during storage in both media. Root dry matter continued to increase in plants stored in 3% sucrose media but did not change in 0% sucrose media. Plants cultured in 3% sucrose media had less leaf chlorosis and less mortality after 7 or 14weeks of low temperature storage than the plantlets from sugar free media. Extending the storage period from 7 to 14weeks or introduction of 2-week dark period at the end of storage did not affect leaf chlorosis or plant mortality during acclimatization. Post-storage growth varied with the cultivar. Benefit of having sucrose in storage media was to develop a strong root system that aided the acclimatization and post-storage growth following 7 or 14week storage. Sucrose loading by culturing plants in liquid media containing 5% sucrose did not allow storage in sugar free media without adversely affecting post-storage growth in both cultivars.     

New expanded bed adsorbents for the recovery of DNA

A 20–40 m pellicular high density (3.7 g cm^–3) expanded bed material has been designed for the capture of DNA and other large macromolecules. Anion exchangers fashioned out of these supports exhibited dramatically enhanced DNA binding capacities over commercial anion exchange adsorbents (6 mg ml^–1, c.f. 50 g ml^–1 at 10% breakthrough), due to a combination of small particle and fuzzy surface architecture created through the coupling of polyethylene imine chains.     

Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis,and astaxanthin synthesis in Escherichia coli

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes -carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, -carotene ketolase (-carotene oxygenase), which converted -carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3S), which was identified after purification by a variety of spectroscopic methods.     

“Costs” of reproduction in reptiles

Summary Many theoretical models of life-history evolution rely on the existence of trade-offs between current fecundity and probable future fecundity and survival. Such costs of reproduction have been demonstrated only rarely. Field and laboratory studies on six species of Australian scincid lizards show that gravid females are more vulnerable to predation than are non-gravid females, primarily because (i) they are physically burdened (running speeds are reduced by 20 to 30%), and (ii) they bask more often (in some species). However, food intake is not reduced in gravid animals. A review of published literature suggests that reproductive trade-offs are widespread among reptiles, but the nature of the reproductive costs may vary widely among related species. Within the range of annual survivorship rates of most lizard species, trade-offs between fecundity and survival are likely to be the main evolutionary determinants of optimal levels of reproductive effort. Trade-offs between fecundity and bodily growth are less likely to be significant.