Comparison of conformational characteristics in structurally similar protein pairs.

Although it is known that three-dimensional structure is well conserved during the evolutionary development of proteins, there have been few studies that consider other parameters apart from divergence of the main-chain coordinates. In this study, we align the structures of 90 pairs of homologous proteins having sequence identities ranging from 5 to 100%. Their structures are compared as a function of sequence identity, including not only consideration of C alpha coordinates but also accessibility, Ooi numbers, secondary structure, and side-chain angles. We discuss how these properties change as the sequences become less similar. This will be of practical use in homology modeling, especially for modeling very distantly related or analogous proteins. We also consider how the average size and number of insertions and deletions vary as sequences diverge. This study presents further quantitative evidence that structure is remarkably well conserved in detail, as well as at the topological level, even when the sequences do not show similarity that is significant statistically.     

Molecular evolution of voltage-sensitive ion channel genes: on the origins of electrical excitability

We have analyzed nucleic acid and amino acid sequence alignments of a variety of voltage-sensitive ion channels, using several methods for phylogenetic tree reconstruction. Ancient duplications within this family gave rise to three distantly related groups, one consisting of the Na+ and Ca++ channels, another the K+ channels, and a third including the cyclic nucleotide-binding channels. A series of gene duplications produced at least seven mammalian homologues of the Drosophila Shaker K+ channel; clones of only three of these genes are available from all three mammalian species examined (mouse, rat, and human), pointing to specific genes that have yet to be recovered in one or another of these species. The Shaw-related K+ channels and the Na+ channel family have also undergone considerable expansion in mammals, relative to flies. These expansions presumably reflect the needs of the high degree of physiological and neuronal complexity of mammals. Analysis of the separate domains of the four-domain channels (Ca++ and Na+) supports their having evolved by two sequential gene duplications and implies the historical existence of a functional two-domain channel.      

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Functional and genetic analysis of annexin VI

This study is concerned with the determination of the function of the 68kDa calcium-binding protein, annexin VI. Studies on the structure and regulation of the gene include a detailed analysis of annexin VI expressed heterologously in human A431 carcinoma cells. We have recently discovered that annexin VI is subject to a novel growth dependent post-translational modification. Interestingly, the protein exerts a negative effect on A431 cells. This effect was manifested as a partial reversal of the transformed phenotype. We are currently exploring the hypothesis that the post-translational modification of annexin VI is required for sub-cellular targeting, and that correct localisation within the cell is essential for function.     

The A34R glycoprotein gene is required for induction of specialized actin-containing microvilli and efficient cell-to-cell transmission of vaccinia virus.

The mechanisms allowing vaccinia virus to spread from cell to cell are incompletely understood. The A34R gene of vaccinia virus encodes a glycoprotein that is localized in the outer membranes of extracellular virions. The small-plaque phenotype of an A34R deletion mutant was similar to that of mutants with deletions in other envelope genes that fail to produce extracellular vaccinia virions. Transmission electron microscopy, however, revealed that the A34R mutant produced numerous extracellular particles that were labeled with antibodies to other outer-envelope proteins and with protein A-colloidal gold. Fluorescence and scanning electron microscopy indicated that expression of the A34R protein was necessary for detection of vaccinia virus-induced actin tails, which provide motility to the intracellular enveloped form of vaccinia virus, and of virus-tipped specialized microvilli that project from the cell. The ability of vaccinia virus-infected cells to form syncytia after a brief exposure to a pH below 6, known as fusion from within, failed to occur in the absence of expression of the A34R protein; nevertheless, purified A34R- virions were capable of mediating low-pH-induced fusion from without. The present study provides genetic and microscopic evidence for the involvement of a specific viral protein in the formation or stability of actin-containing microvilli and for a role of these structures in cell-to-cell spread rather than in formation of extracellular virions.     

Legionella sainthelensi: a new species of Legionella isolated from water near Mt. St. Helens

Six strains of a new species, Legionella sainthelensi, were isolated from freshwater in areas affected by the volcanic eruptions of Mt. St. Helens in the state of Washington. Strains of L. sainthelensi are culturally and biochemically similar to other legionellae. They grow on buffered charcoal yeast agar but not on media that lack cysteine. They are gram-negative, nonsporeforming, motile rods that are positive in reactions for catalase, oxidase, gelatin liquefaction, and beta-lactamase. They are negative in reactions for urease, hydrolysis of hippurate, reduction of nitrates, fermentation of glucose, and blue-white autofluorescence. Their cell wall fatty acid composition is qualitatively similar to those of other legionellae, with 50 to 62% branched-chain fatty acids. They contain the isobranched-chain 14- and 16-carbon acids and anteisobranched-chain 15- and 17-carbon acids and relatively large amounts of straight-chain 16-carbon acid. All strains of L. sainthelensi contain approximately equal amounts of ubiquinones Q9, Q10, Q11, and Q12, a pattern similar to those of Legionella bozemanii, Legionella dumoffi, and Legionella longbeachae. Serological cross-reactions were observed between L. sainthelensi, both serogroups of L. longbeachae, and Legionella oakridgensis. Three strains of L. sainthelensi were greater than 90% related by DNA hybridization. The type strain of L. sainthelensi, Mt. St. Helens 4, was 36% related to the type strain of L. longbeachae and 3 to 14% related to the other nine described Legionella species.     

Streptomycin retards the phenotypic maturation of chick myogenic cells

Summary As part of an effort to optimize conditions required for the complete maturation of muscle cells in vitro, we have investigated the effects of the antibiotics penicillin, streptomycin, and Fungizone (amphotericin B) on the development of cultured chick embryo skeletal muscle. It is shown that even low dosages of streptomycin, but not penicillin or Fungizone, retard protein synthesis and accumulation in these cultures. Myosin accumulation was also reduced and the appearance of striations in fused cells was delayed in myotubes formed in medium containing streptomycin. Additional data suggest that this overall retardation of myogenesis is due to the influence of streptomycin on maturing myotubes rather than early proliferation and cell fusion. These results are discussed with regard to recent efforts to promote the full maturation of muscle cells grown in culture. This research was supported by National Institutes of Health Grant NS 155882 and a Task Force on Drug Development Research Contract from The Muscular Dystrophy Association.     

Ultrasonic characteristics of frozen liver

The recent development of new ultrasound probes has made real-time intraoperative monitoring of cryosurgery, and thermocouple placement a possibility. It is shown that frozen tissue and thermocouple needles have acoustic characteristics that enable them to be easily visualized by ultrasound examination. Further in vivo animal studies are needed to examine temperature characteristics of visualized cryolesions, to develop scanning techniques, and to correlate ultrasonic findings with histologic changes in tissue.     

Utilisation of wild relatives in the genetic improvement of Arachis hypogaea L.

Summary Cross-compatibility of species in section Arachis Krap. et Greg. nom. nud., and chromosome pairing and pollen fertility in their interspecific F₁ hybrids were studied to further understand the phylogenetic relationships among these species. Except those with A. batizocoi Krap. et Greg. nom. nud., hybrids between diploid species have near normal bivalent frequency (9.1–9.8) and moderate to high pollen fertility (60–91%). Hybrids between A. batizocoi and other species have low bivalent frequency (5.2–6.9) and very low pollen fertility (3–7%). These results confirm the earlier separation of these species into two groups based on karyomorphology and Mahalanobis D² calculated on arm ratios. These studies also provide a picture of relative affinities between A. batizocoi, the lone member of one cluster, and the other species, and among the rest of the species. They also indicate that the basic chromosome complement in the two groups of species is the same. Chromosome pairing in triploid hybrids, (A. hypogaea L. X diploid wild species), suggests that A. batizocoi is the closest diploid relative of A. hypogaea. It is closer to A. hypogaea subspecies fastigiata Waldron than to A. hypogaea subspecies hypogaea Krap. et. Rig. Other diploid species of the section Arachis are equidistant from A. hypogaea, and have the same genome which has strong homology to one of the genomes of A. hypogaea. Based on the present results, the two tetraploid species, A. monticola Krap. et Rig. and A. hypogaea can be recognised as two forms of the same species. Breeding implications have been discussed in the light of chromosome behaviour observed in hybrids of A. hypogaea X diploid species, and on the presumptions that A. hypogaea has an AABB genomic constitution, and that among the diploid species, the B genome is present in A. batizocoi while the A genome is common to the other diploid species of section Arachis.Submitted as Journal Article No. 328 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)