Length and interspersion of repetitive and non repetitive DNA sequences in four Amphibian species with different genome sizes

The interspersion period of repetitive and unique sequences was analyzed by two different methods, electron microscopy and agarose gel electrophoresis, for four Amphibian species with different nuclear DNA content, namely the Anura Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) and the Urodela Triturus cristatus (23 pg) and Necturus maculosus (52 pg). Within each of the two subclasses it has been found that interspecific differences, in DNA content, due to variations in the amount of repetitive sequences, do not involve variations in length of the interspersed repetitive sequences. They remain about 380 base pairs. Furthermore, the unique sequences length has been found to be shorter in Bufo (760 base pairs) than in Xenopus (1600) and in Necturus (880) than in Triturus (1340). A study of the interspersion period has shown that the great difference in DNA content between Anura and Urodela, which had been previously shown not to have involved changes in the relative amounts of the various sequence classes, does not involve changes in the interspersion period.     

Adaptative differential maturation of neurohypophyseal provasotocin in amphibians: presence of hydrin 2(vasotocinyl-glycine) in anura but not in urodela]

Occurrence of hydrin 2 (vasotocinyl-glycine), an intermediate in the provasotocin processing, has been shown in the neurointermediate lobe of the pituitary of semi-aquatic or terrestrial amphibian species belonging to the order Anura, namely Rana esculenta, R. temporaria, R. pipiens (Ranidae), and Bufo bufo, B. marinus, B. ictericus (Bufonidae). In contrast hydrin 2 appears absent in species belonging to the order Urodela, namely the axolotl Ambystoma mexicanum (Ambystomidae) and the pleurodele newt, Pleurodela waltlii (Salamandridae). Whereas water transport through the skin and the urinary bladder is stimulated by vasotocin and hydrin 2 in anuran species, this stimulation is known not to occur in urodele species.     

Clustered and interspersed repetitive DNA sequences in four amphibian species with different genome size.

We have compared the amount of clustered and interspersed repetitive sequences in the genome of four Amphibia with different DNA contents per haploid nucleus: two Anura (Xenopus laevis, 3 pg and Bufo bufo, 7 pg) and two Urodela (Triturus cristatus, 23 pg and Necturus maculosus, 52 pg). High molecular weight DNA of the four species was denatured and reassociated to the same Cot in order to obtain duplex sequences with a similar reiteration frequency. Single-stranded DNA was digested off with the Aspergillus S1 nuclease. DNA was then fractionated according to the molecular weight through an agarose A-50 column. We found that the amount of long repetitive sequences is roughly proportional to the genome size in the four species, while the number of short (about 300 base pairs) repetitive sequences is increased many-fold in the species with the larger DNA content, both in Anura and in Urodela.     

Serum proteins and erythrocyte enzymes. Evaluation of frequencies in 2 populations in the Gabon

The results of the electrophoretic phenotyping of two serum groups (C'3 and Transferrine) and seven red cell enzymes (PAc, PGM, AK, ADA, 6PGD, sGPT and Est D) in two groups of negroes from Gabon are presented. The frequencies are in the normal range observed for African populations except for acid phosphatase in Southern Africa; Khoisan people have frequencies of the "Negroe allele R" higher than in any other population of the world. In Obamba and Bateke populations frequencies of Pr is 0.013.     

The determination of ectodermal derivatives in various species of amphibia

The process of labyrinth determination has been studied in amphibians (three Urodela and seven Anura species) using homoplastic transplantation of ear ectoderm, containing labyrinth material onto the abdominal wall of embryos of the same stage of development. The stage of appearance of organ-specific properties in ear ectoderm was determined and the increase of these properties in the course of development was observed. The frequency of ear vesicles formation, the level of their differentiation and their size served as criteria. These criteria allow to align the studied species into a row, where organ-specific properties appear earlier and most completely in representatives of Ranidae family and in Bufo viridis, and weakest, in smooth newt. A comparison of properties of labyrinth material and other areas of ectoderm allowed to conclude that specific differences in determination of different ectodermal primordia are based on specific peculiarities of the whole ectoderm. The appearance of these differences can be explained by the shift in the beginning of gastrulation towards later stages of cleavage during the evolution of amphibians.     

两栖动物线粒体基因组结构特征分析

对2007年6月13日以前公布于GenBank上的78种两栖动物的线粒体基因组全序列进行了总结、比较和分析。78种基因组中基因的数量从35~41个不等;根据基因的数量、种类及其排列顺序的差别将其分为22种基因组类型,其进而聚为3组,其中类型4为两栖纲与其它脊椎动物的常见类型,类型8为两栖纲中现生3个目的公有类型。与类型4比较,其余21种线粒体基因组类型涉及基因变动的基因共有18个,其中变动比较多的是tRNA基因,移位、增多和缺失的发生频率都较大,而蛋白编码基因比较稳定,主要是移位。78种两栖动物中,蚓螈目的线粒体基因组均小于18000bps,多数在15000~16000bps;有尾目和无尾目均大于16000bps,其中有尾目多数在16000~17000bps,无尾目的多数在17000~18000bps。     

Coevolution of the Monogenoidea (Platyhelminthes) based on a revised hypothesis of parasite phylogeny

A revised hypothesis for the phylogeny of the Subclass Polyonchoinea (Monogenoidea) was contructed employing phylogenetic systematics. The Acanthocotylidae (formerly of the Order Capsalidea) is transferred to the Order Gyrodactylidea based on this analysis. The new phylogeny is used to determine coevolutionary relationships of the familial taxa of Monogenoidea with their hosts. The coevolutionary analysis suggests that the Monogenoidea apparently underwent sympatric speciation or dispersal while parasitic on ancestral Guathostomata, resulting in two primary clades: the Polyonchoinea and the Oligonchoinea + Polystomatoinea. The two parasite clades apparently cospeciated independently with divergence of the Chondrichthyes and Osteichthyes. In the Polyonchoinea, the clade associated with Chondrichthyes experienced primary extiaction within the Holocephala, but coevolved into the Loimoidae and Monocotylidae in the Galeomorphii and Squalea (Elasmobranchii), respectively. Within the Osteichthyes, polyonchoineans experienced primary extinction with the divergence of Sarcopterygii, Polypteriformes and Acipenseriformes. They demonstrate primary dispersal from the Neopterygii into the Squalea (as Amphibdellatinea), Actinistia (as Neodactylodiscinea) and Urodela (as Lagarocotylidea). Secondary dispersals of polyonchoineans occurred in the Gyrodactylidae to the Polypteriformes, Urodela and Anura; in the Acanthocotylidae to the Myxinoidea and Squalea; in the Capsalidae to the Acipenseriformes and Elasmobranchii; and in the Monocotylidae to the Helocephala. The Oligonchoinea and Polystomatoinea developed upon divergence of the Chondrichthyes and Osteichthyes. Oligonchoineans cospeciated within the Chondrichthyes, with the Chimaericolidea developing within the Helocephala and the ancestor of the Diclybothriidea + Mazocraeidea within the Elasmobranchii. Two cases of primary dispersal occurred within this clade: the Diclybothriidae to the Acipenseriformes and the ancestor of mazocracidean families to the Neopterygii (both Osteichthyes). Secondary dispersal within the Oligonchoinea includes host switching of the common ancestor of Callorhynchocotyle (Hexabothriidae) to the Holocephala. Polystomatoineans coevolved within the Osteichthyes, but experienced primary extinctions in the Actinopterygii, Actinistia, Dipnoi and Amniota. Coevolution of the Sphyranuridae and Polystomatidae occurred with divergence of the Urodela and Anura, respectively. Secondary dispersal of polystomatids to the Urodela, Dipnoi and Amniota is suggested. A preliminary phylogenetic analysis of the Polystomatoinea suggests that primary extinction with secondary dispersal of polystomatids to the Dipnoi may not be necessary to explain extant parasite distributions, since Concinnocotyla (Concinnocotylinae) appears to represent the sister taxon of the remaining Polystomatidae + Sphyranuridae.     

Visceral pigmentation in four Dendropsophus species (Anura: Hylidae): Occurrence and comparison

Amphibians share with other ectothermic vertebrates an extracutaneous pigmentary system consisting of melanin-containing cells in various organs and tissues. This paper describes the interspecific variation in the visceral pigmentation and extracutaneous pigment system in four species of the genus Dendropsophus [i.e., D. elianeae, D. minutus, D. nanus, and D. sanborni (Anura: Hylidae)]. Fifteen adult males from each species were collected in the region of São José do Rio Preto (State of São Paulo, Brazil), and their visceral pigmentation was analyzed during the reproductive period. The individuals were weighed and measured, and the pigmented visceral cells were classified and documented in photographs. The shape and quantity of the pigment cells differed among the various structures of the same individual, as well as among the same structures of different species. Similarities and differences among these species were observed, and a visceral pigmentation pattern for anatomical structures was detected. In the digestive system of all species analyzed, there was an absence of pigment cells on the stomach and middle intestine. However, the pigmentation of the final portion of the intestine (i.e., the rectum) showed interspecific variation, with D. minutus presenting intense pigmentation, whereas the other species presented no pigmentation. Significant differences were detected also in the cardio-respiratory system, mesentery, and lumbosacral peritoneum.     

Surpellic films, lung surfactant, and their cellular origin in newt, caecilian, and frog

The lung lining and its surfactant have been studied in one species from each of the amphibian sub-classes Anura, Urodela, and Apoda. The surfactant was studied by observation of bubbles derived from the lungs, the morphology by electron microscopy. Other details of the apodan are also given.
The surfactant layers of Rana temporaria and Ichthyophis (species unknown) resemble one another, being somewhat less stable than that of mammals. That of Triturus vulgaris is even less stable, and shows qualitative differences from the other two. Nevertheless, it can reduce the surface tension to 2 mN/m.
The epithelial cells of amphibian lungs cannot be divided into two clearly defined types, as can those of mammals. Microvilli are present on the alveolar surface of all these cells. In the lungs of Rana and Ichthyophis the lining cells contain numerous lamellated osmiophilic bodies (which are intracellular depots of surfactant); in the former their appearance is mainly cross-barred, in the latter mainly concentric. Similar bodies exist in the lung epithelial cells of Triturus but are relatively rare.     

DNA reassociation kinetics in relation to genome size in four amphibian species

DNA reassociation kinetics were studied, by means of the hydroxyapatite chromatography method, for four species of Amphibians with different nuclear DNA content: Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) of the Anura subclass and Trituras cristatus (23 pg) and Necturus maculosus (52 pg) of the Urodela subclass.Within each subclass the two species studied were found to have about the same absolute amount of unique DNA. The differences of total nuclear DNA can be accounted for by quantitative variations of the repetitive sequence classes, at least in part due to changes in the number of copies of the various sequences. On the contrary the great difference in nuclear DNA between the two subclasses, Anura and Urodela, involves all sequence classes in parallel; the slowly reassociating fraction appears to be unique in spite of a tenfold difference in absolute amount.The dependence of reassociation kinetics on DNA fragment length for the four species indicates for all of them an interspersed organization of the various sequence classes.     

The chromophore structures of the Pr States in plant and bacterial phytochromes

The resonance Raman spectra of the Pr state of the N-terminal 65-kDa fragment of plant phytochrome phyA have been measured and analyzed in terms of the configuration and conformation of the tetrapyrroles methine bridges. Spectra were obtained from phyA adducts reconstituted with the natural chromophore phytochromobilin as well as phycocyanobilin and its isotopomers labeled at the terminal methine bridges through (13)C/(12)C and D/H substitution. Upon comparing the resonance Raman spectra of the various phyA adducts, it was possible to identify the bands that originate from normal modes dominated by the stretching coordinates of the terminal methine bridges A-B and C-D. Quantum chemical calculations of the isolated tetrapyrroles reveal that these modes are sensitive indicators for the methine bridge configuration and conformation. For all phyA adducts, the experimental spectra of Pr including this marker band region are well reproduced by the calculated spectra obtained for the ZZZasa configuration. In contrast, there are substantial discrepancies between the experimental spectra and the spectra calculated for the ZZZssa configuration, which has been previously shown to be the chromophore geometry in the Pr state of the bacterial, biliverdin-binding phytochrome from Deinococcus radiodurans (Wagner, J. R., J. S. Brunzelle, K. T. Forest, R. D. Vierstra. 2005. Nature. 438:325-331). The results of this work, therefore, suggest that plant and bacterial (biliverdin-binding) phytochromes exhibit different structures in the parent state although the mechanism of the photoinduced reaction cycle may be quite similar.     

Tadpole Xenopus laevis hemoglobin. Correlation between structure and functional properties

Perutz & Brunori (1982) proposed that the COOH-terminal His and Ser F9 of the beta-chains of fish and amphibian hemoglobins are responsible for their Root effect and part of their alkaline Bohr effect. Analysis of the kinetics of carbon monoxide binding by hemoglobin from the tadpole of Xenopus laevis supports that model and suggests an explanation for the absence of an alkaline Bohr effect in many aquatic Anura and Urodela.     

Saccharomyces cerevisiae Est3p dimerizes in vitro and dimerization contributes to efficient telomere replication in vivo

In Saccharomyces cerevisiae at least five genes, EST1, EST2, EST3, TLC1 and CDC13, are required for telomerase activity in vivo. The telomerase catalytic subunit Est2p and telomerase RNA subunit Tlc1 constitute the telomerase core enzyme. Est1p and Est3p are the other subunits of telomerase holoenzyme. In order to dissect the function of Est3p in telomere replication, we over-expressed and purified recombinant wild-type and mutant Est3 proteins. The wild-type protein, as well as the K71A, E104A and T115A mutants were able to dimerize in vitro, while the Est3p-D49A, -K68A or -D166A mutant showed reduced ability to dimerize. Mutations in Est3p that decreased dimerization also appeared to cause telomere shortening in vivo. Double point mutation of Est3p-D49A-K68A and single point mutation of Est3p-K68A showed similar telomere shortening, suggesting that the K68 residue might be more important for telomerase activity. The ectopic co-expression of K71A or T115A mutant with wild-type Est3p using centromere plasmids caused telomere shortening, while co-expression of the D49A, K68A, D86A or F103A mutants with wild-type Est3p had no effect on telomere length regulation. These data suggested that dimerization is important for Est3p function in vivo.     

Comparative morphology of the livers in amphibians]

Amphibians are divided into three orders: Gymnophiona, Urodela and Anura. We studied on the correlation between the liver structures in the three orders of amphibians by histological technique. Livers of thirty species of amphibian were fixed by perfusion with paraformaldehyde, and observed by light microscopy in special staining for elastic fibers. The cytoskeletal components of the hepatic stellate cells and myofibroblasts were identified by immunohistochemistry for glial fibrillary acidic protein and a-smooth muscle actin. Among the three orders of amphibians, Gymnophiona and Urodela were characterized the development of lymph vessels in liver capsules and Glisson's sheath. The present study indicates that there are differences in the pattern of hepatic histological components in the amphibians examined at this time. We discussed their diverged mode of living that can be categorized into three groups: aquatic, aqua-terrestrial and fully terrestrial group.     

Primary teeth in Anura are nonpedicellate and bladed

1. Scanning electron microscop of the dentition of four anuran species (Discoglossus pictus, Bombina orientalis, Rana cyanophlyctis, Rana temporaria) revealed that at least the primary teeth, which are established during metamorphosis, are more or less bicuspid, bladed and nonpedicel-late. Juveniles and adults, however, have pedicellate teeth. 2. Thus, regardin the pedicellate condition, the sequence of stages in tooth development of Anura und Urodela show some similarities, although the primary teeth of urodeles develop already before metamorphosis.     

Analysis of linkage between biochemical and morphological markers of 1R-, 2R-, and 5R-chromosomes in rye with autofertility mutations in main incompatibility loci

In F2 hybrids between self-sterile plants of the Volkhova cultivar and self-fertile lines with established self-fertility mutations (sf-mutations) at the major incompatibility loci S (1R), Z (2R), and T (5R), the effect of sf-mutations on the inheritance of secalin-encoding, isozyme, and morphological markers located on the same chromosomes was investigated. Linkage between loci Prx7 and S and locus Sec3 coding for high-molecular-weight secalins on chromosome 1R was shown for the first time The frequency of recombination between Prx7 and Sec3 and between S and Sec3 was 29.1 +/- 4.8% and 30.9 +/- 7.0%, respectively. Independent inheritance of locus Z and isozyme markers of chromosome 2R, Est3/5 and beta-Glu, from locus Sec2 encoding 75-kDa gamma-secalins was shown; in hybrids, the recombination frequency between Est3/5 and locus Z varied from 19.2 +/- 8.1 to 50%. Independent inheritance of morphological (Ddw and Hs) and isozyme markers (Est4, Est6/9, and Aco2) of chromosome 5R from locus T located on the same chromosome was demonstrated.     

Genetic control of leucine aminopeptidase and esterase isozymes in the interspecific cross Cucurbita ecuadorensis x C. maxima

Starch gel electrophoretic analyses of crude seed extracts of Cucurbita ecuadorensis, C. maxima, their F₁ and F₂, and three of the four possible interspecific backcrosses reveal that the genus is polymorphic for alpha-naphthyl acetate esterases (Est) and leucine aminopeptidase (LAP). The two electrophoretic forms of both Est and LAP are controlled by codominant alleles. The two loci do not exhibit linkage. Neither the LAP nor the Est phenotypes exhibit a significant deviation from the expected 1:1 ratio in interspecific backcrosses when the donor parent alleles are transmitted through female gametes, but there is a significant deviation for Est when transmission is through male gametes. Differential gametic selection involving the Est-1 locus suggests structural differences between the genomes of the parental species for the chromosomal region in which this locus occurs. No structural differences are indicated between the parental genomes for the chromosome region bearing the Lap-1 locus.     

pH-dependent spectroscopic changes associated with the hydroquinone of FMN in flavodoxins

Photoreduction with a 5-deazaflavin as the catalyst was used to convert flavodoxins from Desulfovibrio vulgaris, Megasphaera elsdenii, Anabaena PCC 7119, and Azotobacter vinelandii to their hydroquinone forms. The optical spectra of the fully reduced flavodoxins were found to vary with pH in the pH range of 5.0-8.5. The changes correspond to apparent pKa values of 6.5 and 5.8 for flavodoxins from D. vulgaris and M. elsdenii, respectively, values that are similar to the apparent pKa values reported earlier from the effects of pH on the redox potential for the semiquinone-hydroquinone couples of these two proteins (7 and 5.8, respectively). The changes in the spectra resemble those occurring with the free two-electron-reduced flavin for which the pKa is 6.7, but they are red-shifted compared with those of the free flavin. The optical changes occurring with flavodoxins from D. vulgaris and A. vinelandii flavodoxins are larger than those of free reduced FMN. The absorbance of the free and bound flavin increases in the region of 370-390 nm (Delta epsilon = 1-1.8 mM-1 cm-1) with increases of pH. Qualitatively similar pH-dependent changes occur when FMN in D. vulgaris flavodoxin is replaced by iso-FMN, and in the following mutants of D. vulgaris flavodoxin in which the residues mutated are close to the isoalloxazine of the bound flavin: D95A, D95E, D95A/D127A, W60A, Y98S, W60M/Y98W, S96R, and G61A. The 13C NMR spectrum of reduced D. vulgaris [2,4a-13C2]FMN flavodoxin shows two peaks. The peak due to C(4a) is unaffected by pH, but the peak due to C(2) broadens with decreasing pH; the apparent pKa for the change is 6.2. It is concluded that a decrease in pH induces a change in the electronic structure of the reduced flavin due to a change in the ionization state of the flavin, a change in the polarization of the flavin environment, a change in the hydrogen-bonding network around the flavin, and/or possibly a change in the bend along the N(5)-N(10) axis of the flavin. A change in the ionization state of the flavin is the simplest explanation, with the site of protonation differing from that of free FMNH-. The pH effect is unlikely to result from protonation of D95 or D127, the negatively charged amino acids closest to the flavin of D. vulgaris flavodoxin, because the optical changes observed with alanine mutants at these positions are similar to those occurring with the wild-type protein.     

Morphogenetic approach to the formation of paired limbs in the course of tetrapodization

Transition from sarcopterygians to tetrapods is analyzed based on new paleontological, ontogenetic, and molecular data. It is shown that transformation of skeletal fin elements into the tetrapod limb followed the patterns of divergent, parallel, and mosaic development. Morphogenetic plasticity and autonomy of these processes as well as the same developmental bauplan for the limbs of Urodela and Anura are proposed. Variations observed in these processes are regarded as a result of larval adaptations and heterochronies. The latter excludes recapitulation of successive archetypical states (transformation-development of the fish fin into tetrapod limb). The idea that the digits are a novelty relative to the distal radials of the fin is supported.     

Direct development in the tailed amphibians (salamanders), its origin and evolution

Biphasic life circle including water larvae and terrestrial adults is the primitive character for recent amphibians. Respectively, the larval development type (sensu Zakhvatkin, 1975) is also primitive for them. However, many amphibians possess direct development, in which the most part of ontogenesis takes place in the egg, and a miniature copy of the adult adapted to the terrestrial mode of life comes into the world. Transition from the larval type to the direct one occurs several times independently in Apoda, Urodela, and Anura. Pathways and mechanisms of formation of the direct type, its evolutionary tendencies, and ontogenetic prerequisites of that transition are studied in the plethodontid urodelans herewith in details. It is observed that the entire process of initial formation and subsequent specialization of the direct type involves: 1) progressing displacement of ontogenesis into embryogenesis and loss of larval characters, 2) desynchronization of initially metamorphic transformations and processes with their progressive lost, 3) acceleration of the beginning of functional activity of the thyroid gland, and 4) subsiding of the role of thyroid hormones in the ontogenesis regulation. Transition to the direct development type involves similar kinds of ontogenetic transformations and regulations in both Anura and Urodela despite of their independent evolution. Respectively, mechanisms of that transition are universal for the amphibians. The ontogenetic prerequisites of that transition are shown to be either significant dissociability of the larval and adult stages of ontogenesis (in anurans and plethodontid urodelans), or absence of the extreme larval specializations and respective sharp differences between larvae and adults (in extant Apoda and extinct labyrinthodonts and seymouriamorphs).