A BC200-derived element and Z-DNA as structural markers in annexin I genes: Relevance to Alu evolution and annexin tetrad formation

We have identified two types of structural elements in genomic DNA for annexin I that provide physical evidence of genetic events leading to conserved changes in gene structure. The sequence upstream of the transcribed region in human annexin I contained a rare, Alu-like repetitive element with flanking direct repeats, probably derived from the active BC200 gene via germline retroposition. Nucleotide substitutions in this BC200 insert relative to the 7SL gene and its absence in rodent annexins I identified it as a recent primate pseudogene. Phylogenetic analysis showed that the BC200 gene represents a new clade of primate Alu evolution that branched near the time of appearance of the progenitor to the free left Alu monomer, FLAM-C. Separate analysis identified a Z-DNA motif in pigeon annexin I intron 7 that may represent the vestigial recombination site involved in primordial assembly of the annexin tetrad. These distinct structural features in annexin I genes provide insight into the evolution of Alu repeats and the mechanism of annexin tetrad formation.     

Preparation,characterization, and structure of half gap junctional layers split with urea and EGTA

Gap junctions, collections of membrane channels responsible for intercellular communication, contain two paired hemichannels (also called connexons). We have investigated conditions for splitting the membrane pair using urea. We have developed a protocol which consistently splits the gap junction samples with 60–90% efficiency. Our results indicate that hydrophobic forces are important in holding the two connexons together but that Ca²⁺ ions are also important in the assembly of the membrane pair. Greater yields and better structural integrity of split junctions were obtained with a starting preparation of gap junctions which had been detergent treated. Image analysis of edge views of single connexon layers reveal an asymmetry in the appearance of the cytoplasmic and extracellular surface. Cryo-electron microscopy and image analysis of split junctions show that the packing and structural detail of membranes containing arrays of single connexons are the same as for intact junctions, and that the urea treatment causes no gross structural changes in the connexon assembly.The antibodies used to analyze the protein composition in our samples were the generous gift of Dr. David Paul. We thank Dr. Camillo Peracchia for sending us a preprint of his chapter on molecular mechanisms of connexon gating and docking which helped guide our thinking about interconnexon forces and John Badger for information on divalent cation sites in protein structure. We thank Amani Thomas-Yusuf for technical assistance. The photographic expertise of Marie Craig is gratefully acknowledged. This work was funded by National Institutes of Health GM43217 to G.E.S. and GM18974 to D.A.G.     

Sequence analysis of the complete mitochondrial DNA molecule of the hedgehog,Erinaceus europaeus,and the phylogenetic position of the Lipotyphla

The sequence of the mitochondrial DNA (mtDNA) molecule of the European hedgehog (Erinaceus europaeus) was determined. The length of the sequence presented is 17,442 nucleotides (nt). The molecule is thus the largest eutherian mtDNA molecule so far reported. The organization of the molecule conforms with that of other eutherians, but the control region of the molecule is exceptionally long, 1,988 nt, due to the presence of repeated motifs at two different positions in the 3 part of the control region. The length of the control region is not absolute due to pronounced heteroplasmy caused by variable numbers of the motif TACGCA in one of the repetitive regions. The sequence presented includes 46 repeats of this type. The other repeated region is composed of different AT-rich repeats. This region was identical among four clones studied. Comparison of mitochondrial peptide-coding genes identified a separate position of the hedgehog among several mammalian orders. The concatenated protein sequence of the 13 peptide-coding genes was used in a phylogenetic study using the opossum as outgroup. The position of the hedgehog sequence was basal among the other eutherian sequences included: human, rat, mouse, cow, blue whale, harbor seal, and horse. The analysis did not resolve the relationship among carnivores, perissodactyls, and artiodactyls/cetaceans, suggesting a closer relationship among these orders than acknowledged by classical approaches. Correspondence to: U. Arnason     

The complete nucleotide sequence of the mitochondrial DNA genome of the rainbow trout,Oncorhynchus mykiss

The complete nucleotide sequence of the mitochondrial DNA of the rainbow trout, Onchorynchus mykiss, has been determined. The total length of the molecule is 16,660 bp. The rainbow trout mitochondrial DNA has the same organization described in eutherian mammals, the clawed frog (Xenopus laevis), and the two fish species, Oriental stream loach (Crossotoma lacustre) and carp (Cyprinus carpio). Alignment and comparison of the deduced amino acid sequences of the 13 proteins encoded by rainbow trout and other vertebrate mitochondrial genomes allowed us to estimate that COI is the most conserved mitochondrial subunit (amino acid identity ranging from 85.6% to 94.8%) whereas ATPase 8 is the most variable one (amino acid identity ranging from 30.8% to 70.4%). Putative secondary structures for the 22 tRNAs found in the molecule are given along with an extensive comparison of tRNA sequences among representative species of each major group of vertebrates. In this sense, an unusual cloverleaf structure for the tRNA^Ser(AGY) is proposed. A stem-loop structure inferred for the origin of the L-strand replication (O_L) and the presence of a large polycytidine tract in the O_L loop is described. The existence of this stretch instead of the usual T-rich sequence reported so far in mammal mtDNAs is explained in terms of a less-strict template dependence of the RNA primase involved in the initiation of L-strand replication. Correspondence to: J.M. Bautista     

陆地棉的核型模式

陆地棉的核型公式为2n=4x=52=30m+20sm(4SAT)+2st(2SAT); 染色体绝对长度平均3.40μm; 臂比值最大与最小的分别是4.02和1.14,平均1.77;随体3对,分别位于第12、19和26对同源染色体的短臂上;最长与最短染色体的比值为2.36;其核型属于Stebbin的2B类型。 Abstract:Karyotype formula of Gossypium hirsutum L.was 2n=4s=52=30m+20m (4SAT)+2st (2SAT); and its absolute lengths of chromosomes averaged 3.40μm; arm ratio differed from 1.24 to 4.02,with the mean value of 1.77; three pairs of satellite chromosomes; the ratio of the longest to the shortest chromosomes was 2.36; and the karyotype belonged to 2B in Stebbin’s classification.     

Conformational studies of a benzodiazepine-like peptide in SDS micelles by circular dichroism, 1H NMR and molecular dynamics simulation

The conformation of a benzodiazepine-like decapeptide corresponding tothe YLGYLEQLLR fragment of a casein has been examined in a sodium dodecylsulfate micellar medium using circular dichroism, two-dimensional¹H NMR spectroscopy and restrained molecular dynamicssimulation. The decapeptide adopts an amphipathic 3₁₀-helicoidstructure in which theE⁶···R¹⁰ ionic bridgestabilizes the C-terminus.     

Cloning of cDNA for Vitellogenin of the Parasitoid Wasp, Pimpla nipponica (Hymenoptera: Apocrita: Ichneumonidae): Vitellogenin Primary Structure and Evolutionary Considerations

The cDNA for vitellogenin (Vg) of the parasitoid wasp Pimpla nipponica (Hymenoptera: Apocrita) was cloned and sequenced.¹ The deduced amino acid sequence with 1807 residues was obtained. The N-terminal 20 amino acids chemically determined for vitellin (Vn) agreed completely with the deduced 20 amino acids that follow the 16 amino acid residues for putative signal peptide. The cDNA clone for the Vg of the turnip sawfly Athalia rosae (Hymenoptera: Symphyta), previously obtained and partially sequenced, was also completely sequenced and the amino acid sequence deduced. Amino acid sequences were compared between these two species and also with known Vg sequences from other insects. Common to all these insects is the presence of two long regions with relatively well-conserved amino acid sequences, one near the N-terminal extending 267–282 residues (including two cysteines at conserved locations), and the other starting at position 450 to 655 and extending 279–283 residues, and of a region at the C-terminal extending some 200 residues (about 250 in Aedes aegypti due to the presence of a serine-rich stretch) with 10 cysteines at conserved locations. A molecular phylogenetic tree was constructed.     

Molecular Evolutionary Analysis of the Thiamine-Diphosphate-Dependent Enzyme,Transketolase

Members of the transketolase group of thiamine-diphosphate-dependent enzymes from 17 different organisms including mammals, yeast, bacteria, and plants have been used for phylogenetic reconstruction. Alignment of the amino acid and DNA sequences for 21 transketolase enzymes and one putative transketolase reveals a number of highly conserved regions and invariant residues that are of predicted importance for enzyme activity, based on the crystal structure of yeast transketolase. One particular sequence of 36 residues has some similarities to the nucleotide-binding motif and we designate it as the transketolase motif. We report further evidence that the recP protein from Streptococcus pneumoniae might be a transketolase and we list a number of invariant residues which might be involved in substrate binding. Phylogenies derived from the nucleotide and the amino acid sequences by various methods show a conventional clustering for mammalian, plant, and gram-negative bacterial transketolases. The branching order of the gram-positive bacteria could not be inferred reliably. The formaldehyde transketolase (sometimes known as dihydroxyacetone synthase) of the yeast Hansenula polymorpha appears to be orthologous to the mammalian enzymes but paralogous to the other yeast transketolases. The occurrence of more than one transketolase gene in some organisms is consistent with several gene duplications. The high degree of similarity in functionally important residues and the fact that the same kinetic mechanism is applicable to all characterized transketolase enzymes is consistent with the proposition that they are all derived from one common ancestral gene. Transketolase appears to be an ancient enzyme that has evolved slowly and might serve as a model for a molecular clock, at least within the mammalian clade. Received: 13 September 1995 / Accepted: 14 November 1996