Molecular cloning and sequencing of mRNAs coding for minor adult globin polypeptides of Xenopus laevis.

Globin mRNA was isolated from immature red blood cells of an adult Xenopus laevis female. mRNA/cDNA hybrids were integrated in the Pst I cleavage site of pBR 322 by G/C tailing, and cloned in Escherichia coli strain HB 101. By restriction site analysis as well as hybridization behaviour we identified two clones coding for minor adult alpha and beta globin chains. Nucleotide sequence analysis and derived amino acid sequences are presented.     

Cloning of the newt Pleurodeles waltlii chromosomal DNA

Pleurodeles waltlii genomic DNA has been cloned using several phage lambda vectors. We have isolated approx. 600 000 clones, which correspond to about 20% of the total DNA sequences of this organism. This constitutes the first large gene library of a Urodele. The low yield of cloning was attributable to the abundance of highly repetitive sequences, since recombinations in the bacterial host could lead to the loss of clones. Indeed, the existence of highly repetitive sequences was directly demonstrated by hybridization between recombinants and the total genome, and some of the cloned DNA was found to be unstable. We suggest new methods for cloning the highly repetitive sequences.     

Effect of thyroid hormones on the switch from larval to adult hemoglobin synthesis in the salamander Pleurodeles waltlii

The evolution of globin chain synthesis was studied in larvae treated either with thyroxine or with an antithyroid substance. Thyroxine treatment accelerated the rate of Hb switching; it induced a preferential synthesis of adult globins while the synthesis of larval globins decreased rapidly. Treatment with thiourea did not prevent the Hb transition, which occurred even at concentrations of thyroid hormones that did not permit induction of anatomical metamorphosis.     

高原世居藏族α、β珠蛋白编码基因的克隆与测序

目的:通过对高原世居藏族α、β珠蛋白编码基因的分析,探讨藏族Hb高氧亲合力的分子机制.方法:高原现场采集健康成年男性藏族人骨髓样品,提取总RNA,通过逆转录聚合酶链反应(RT-PCR)获得人α和β珠蛋白的cDNA,与PGEM-T Easy质粒连接后,将α和β珠蛋白的cDNA转化JM109大肠杆菌中扩增培养,经酶切鉴定后测序,结果与NCBI数据库进行同源性比较.结果:藏族人α珠蛋白的cDNA与NCBI数据库登录的人cDNA序列相同,没有突变位点.一例藏族人β珠蛋白143位密码子发生了氧亲和力增高的碱基突变(CAC-＞CGC),其对应的氨基酸由His变为Arg(即Hb Abruzzo).结论:藏族人高氧亲和力变种的发现,为今后高原低氧适应相关基因的研究提供了线索.     

Cloning of two adult hamster globin cDNAs (alpha and beta major).

A cDNA library was prepared from poly(A) mRNA extracted from adult anemic hamster spleen erythroid cells. cDNA clones containing inserts coding for adult alpha and beta major globin chains were isolated. Their identity was confirmed by (a) translation of hybrid selected mRNA and (b) nucleotide sequence analysis of the inserts and comparison to the adult globin cDNAs of mouse, rabbit and human. Availability of sequences for embryonic (Li et al. (1992) Biochim. Biophys. Acta 1130, 218-220) and adult globin cDNAs (this report) will aid in investigations of the molecular mechanisms involved in the globin ontogeny of hamsters.     

Morphological,histochemical, and ultrastructural characterization of the accessory cells of neuromasts in the salamander Pleurodeles waltlii

Summary The ultrastructural and histochemical features of the accessory cells of the neuromast of the salamander P. waltlii have been examined. Three types of accessory cells, supporting, mantle, and basal, were found, but only the first 2 are considered in this article. Supporting cells characterized by a highly dilated endoplasmic reticulum occur among and surrounding sensory cells. Mantle cells, morphologically different from the supporting cells, surround the remainder of the neuromast. Both types of accessory cells exhibit histochemically different secretory materials. Our morphological results suggest that both accessory cells contribute to the formation of cupular material.     

The characteristics of osteoclast localization in the bones of the extremities of the crested salamander Pleurodeles waltlii]

Localization of polynuclear osteoclasts in limb bones of ribbed newt were analyzed in consideration of calcium content in different bone structures. With using the methods of light and scanning electron microscopy and X-ray microanalysis it was shown that a critical level of cartilage calcification is need to activate osteoclasts. Osseous tissue can bind more calcium salt without resorption.     

The development of the osteocranium of Pleurodeles waltlii Michahelles

Under optimal conditions the development of the osteocranium covers the major part of the larval period and lasts about three and a half months. The earliest ossifications are of dermal origin and concern the tooth-bearing bones. The poorly developed enchondral ossifications found in Urodela, appear at a much later stage, just before metamorphosis.     

Fibronectin synthesis during oogenesis and early development of the amphibian Pleurodeles waltlii

Fibronectin (FN) synthesis during oogenesis and early embryogenesis of the amphibian Pleurodeles waltlii was investigated. The isotopically labelled amino acids [3H]leucine and [35S]methionine were incubated with oocytes or microinjected into embryos. Newly synthesized FN was analysed by polyacrylamide gel electrophoresis, using the high resolution two-dimensional gel system described by O'Farrell. With this method and fluorography we demonstrate that FN synthesis begins during oogenesis. De novo synthesized FN appears during cleavage and gastrulation. Using actinomycin D we show the presence of maternal messenger RNA coding for FN. It is translated during the cleavage and gastrulation stages.     

Experimental gynogenesis in the newt species Pleurodeles waltlii and P. poireti

Eggs of diploid females of Pleurodeles waltlii, inseminated by genetically inactivated sperm of Salamandra salamandra, have been treated by heatshock or increased hydrostatic pressure during the first hour of their development. The resulting viable gynogenetic individuals show different degrees of ploidy although they mostly are diploid. The use of females with a pericentric inversion as a marker chromosome allow the chromosomal constitution to be clarified. Eggs of females heterozygous for a recessive semilethal mutation (ascite caudale, ac) subjected to the same experimental treatment gave 50% ac/ac embryos. Experiments with Pleurodeles poireti gave the same results as those with P. waltlii. These observations prove that gynogenetic Urodeles can be produced in large numbers. In their offspring the detection of inherent or spontaneous recessive mutations is greatly facilitated.

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Sommaire Des oeufs de femelles diploïdes de Pleurodeles wahlii sont inséminés par du sperme de Salamandra salamandra inactivé du point de vue génétique. Ils sont soumis pendant la première heure de développement soit à une élévation de température (37°1 pendant 5 minutes), soit à une compression (450 bars pendant 6 minutes). On obtient des individus gynogénétiques viables de ploïdie variable mais en majorité diploïdes. L'utilisation de femelles diploïdes de P. waltlii présentant un chromosome marqueur permet de démontrer l'origine gynogénétique des descendants et de préciser la manière dont s'est constitué leur équipement chromosomique. Des oeufs de femelles diploïdes de P. waltlii hétérozygotes pour la mutation récessive semi-léthale ascite caudale, inséminés et choqués selon le même protocole conduisent à 50% d'embryons gynogénétiques homozygotes pour cette mutation. La même technique appliquée à des oeufs de femelles diploïdes de Pleurodeles poireti permet d'obtenir également des individus gynogénétiques, de ploïdie variable. Ces résultats confirment la possibilité d'obtenir en grand nombre des individus gynogénétiques viables chez les Urodèles. Chez les individus hétérozygotes pour un gène récessif, la détection et l'analyse des mutations spontanées ou induites peuvent être grandement facilitées par ce mode de reproduction.

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The authors would like to dedicate this article to the memory of the late Jean Rostand (1894–1977) pioneer of experimental diploid gynogenesis in amphibia     

Cloning and sequencing of two genes fromStaphylococcus carnosus coding for glucose-specific PTS and their expression inEscherichia coliK-12

Phosphoenolpyruvate (PEP)-dependent phosphorylation experiments have indicated that the grampositive bacteriumStaphylococcus carnosus possesses an EIICBA fusion protein specific for glucose. Here we report the cloning of a 7 kb genomic DNA fragment containing two genes,glcA andglcB, coding for the glucose-specific PTS transporters EII^Glc1 and EII^Glc2 which are 69% identical. The translation products derived from the nucleotide sequence consist of 675 and 692 amino acid residues and have calculated molecular weights of 73 025 and 75 256, respectively. Both genes can be stably maintained inEscherichia coli cells and restore the ability to ferment glucose toptsG deletion mutants ofE. coli. This demonstrates the ability of the PTS proteins HPr and/or EIIA^Glc of a gram-negative organism (E. coli) to phosphorylate an EIICBA^Glc from a gram-positive organism (S. carnosus).     

Cloning and sequencing of an Aspergillus niger gene coding for β-fructofuranosidase

A 1.7 kb DNA fragment encoding b-D-fructofuranosidase in a strain of Aspergillus niger was amplified by PCR, inserted into a 4.4 kb cloning vector and sequenced. Comparison of this sequence with suc1 (GenBank) showed that this invertase gene had suffered a single base deletion followed by a single base insertion 49bp further down-stream with the result that 16 amino acids are coded by a different reading frame.