Selenocysteine's mechanism of incorporation and evolution revealed in cDNAs of three glutathione peroxidases

The nonsense codon, UGA, has for the first time recently been shown to encode selenocysteine in two proteins, mouse glutathione peroxidase (GSH-Px) (EC 1.11.1.9) and bacterial formate dehydrogenase. A co-translational rather than post-translational selenium-incorporation mechanism has been implicated. Furthermore, high expression levels of GSH-Px have suggested that suppression of termination is efficient and specific. We have isolated and characterized pituitary, kidney and placenta cDNAs for bovine, human and mouse GSH-Px respectively. It is demonstrated that this novel suppression event occurs in diverse tissues, in at least three mammalian species and at the translational step. Surprisingly, GSH-Px is shown to be extramitochondrially encoded, indicating a cytosolic suppression event rather than one utilizing the mitochondria's well-documented extended codon-reading ability. Sequence analysis reveals that a simple proximal contextual pattern responsible for readthrough does not exist. Analysis of predicted secondary structures of mRNAs, however, has revealed a conformation which may be unique to selenocysteine proteins and may prove useful as a tool for artificial incorporation of selenocysteines. A human intron for GSH-Px from an unspliced mRNA has been isolated whose position indicates an ancient, divergent evolutionary relationship with thioredoxin-S2, rather than an independent convergent one.     

Decreased incidence of mycorrhizal root tips associated with soil heavy-metal enrichment

Mycorrhizal incidence was studied at two forested locations in south-central Virgina. At each location, one site with soil naturally enriched in copper, lead and zinc was designated as mineralized, and an adjacent site, with significantly lower levels of these metals, was used as a control. A total of 223 soil samples were collected during the summer of 1984 and assayed for active mycorrhizal tips. A reduced active mycorrhizal root tip count was found in those samples collected from the mineralized sites at both locations (P≤0.001).     

A continuous noncontact method for measuring in situ vascular diameter with a video camera

A new, continuous, on-line, video diameter-measuring technique, utilizing a video camera mounted on the sidearm of a stereo microscope, is described. Vessel diameter is derived from changes in the video output signal of the camera or a video recorder when the vessel of interest is displayed horizontally on a monitor and well contrasted with its background. A comparator threshold is set on the filtered video output signal and generates an output pulse that is used to gate horizontal video sync pulses to a digital counter-timer. The number of pulses counted for each video field (no. of horizontal video lines) is proportional to the vessel diameter. The video-derived diameter is calibrated using known standards and correlates well with sonomicrometer-derived diameters of the carotid artery and jugular vein during increasing pressure ramps (r greater than 0.999). The diameter update rate is 60 Hz, and the resolution of the system is one horizontal video line, independent of the vessel size. With suitable magnification and contrast both arteries and veins as small as 200 micron have been measured using this system.     

Identification of a CA repeat at the TCRA locus using yeast artificial chromosomes: a general method for generating highly polymorphic markers at chosen loci.

The creation of a comprehensive genetic map in human has been limited by the lack of highly polymorphic markers spaced evenly throughout the human genome. We have utilized yeast artificial chromosomes (YAC) containing large human DNA inserts to help identify highly polymorphic (CA)n repeats at a chosen locus. The DNA of a YAC containing the locus was subcloned in M13 vectors, and the recombinants were screened at high stringency to detect preferentially long (CA)n repeats (n greater than 20). These repeats, which are the most likely to be highly polymorphic, were then studied to confirm both the level of polymorphism and their precise genetic location. This strategy has permitted the identification of a new, highly polymorphic CA repeat (77% heterozygosity) at the T cell receptor alpha chain (TCRA) locus on chromosome 14q. It provides a powerful marker for assessing the role of this locus in the susceptibility to autoimmune and infectious diseases. This approach should permit the development of highly polymorphic markers at any targeted locus and rapidly improve the current human genetic map.     

The problem of movelength and turn definition in analysis of orientation data.

This study examines the analysis of arthropod orientation data. Three problems are discussed: (1) dealing with time as it applies to spatial data, (2) determining the appropriate movelength to be used in collecting and in analyzing data, and (3) defining a turn, to discriminate between "gait noise" and course changes. The main objective is to determine the solution to defining the most appropriate movelength for comparisons between variables and between species. The technique described here for selecting the appropriate movelength that has relevance to both the locomotory rate of the animal and its body length, reduces variation resulting from lateral translational movements, prevents the use of movelengths that lead to artifactual or unrealistic turning values per move, and permits comparisons of species and individuals under various stimulus conditions.     

DNA sequence analysis of revertants of the hisD3052 allele of Salmonella typhimurium TA98 using the polymerase chain reaction and direct sequencing: application to 1-nitropyrene-induced revertants

We have used the polymerase chain reaction (PCR) to speed the DNA sequence analysis of revertants of Salmonella typhimurium TA98. Briefly, a crude DNA extract from a single colony was prepared and used in an asymmetric PCR to amplify a 328-bp fragment containing the hisD3052 mutation approximately in the center. Following ultrafiltration, the ssDNA was sequenced using an end-labeled probe and dideoxy sequencing. The most frequent mutation among the revertants was a -2 deletion of GC or CG within the sequence CGCGCGCG, which is upstream of the hisD3052 mutation. This deletion occurred in 38% (6/16) of the spontaneous (-S9) revertants and in 94% (15/16) of a set of 1-nitropyrene-induced revertants. Other mutations, mostly deletions but also some complex mutations (i.e., single mutational events involving a combination of insertions, deletions, and substitutions), occurred within quasipalindromic regions of DNA. Possible mutational mechanisms are discussed, and the results with 1-NP are compared to those obtained in other systems.     

Recipes for reconstituting skin

Reconstituted Living Skin Equivalent (LSE) is made up of a dermal equivalent (DE) on which keratinocytes are plated where they give rise to a multilayered differentiated epidermis. The dermal equivalent develops through interactions between fibroblasts and collagen fibrils that begin to form after the cell-matrix precursor is cast. The gel that forms as a result of collagen polymerization and fluid trapping is contracted uniformly in all dimensions. By securing it at ends and edges in the mold in which it is cast, the final dimensions, strength and morphology of the forming tissue are altered. The same phenomena are seen in casting tubular tissues for the fabrication of small caliber blood vessel equivalents. The cells of the dermal equivalent are biosynthetically active and enrich the matrix to different degrees with secretory products, depending on how the cells are stimulated and on the presence or absence of an epidermis. Collagen biosynthesis by dermal cells in the DE is sensitive to growth factors, ascorbate concentrations and amino acid pools. Both ascorbate and TGF beta 1 increase total collagen biosynthesis at least two-fold by one week after tissue formation. With TGF beta 1 present, the capacity of cells in the DE to synthesize collagen increases with time, over a two-week period. If ascorbate (200 micrograms/ml) is added just after the tissue is cast and daily thereafter, contraction lattice is blocked, and collagen biosynthesis is enhanced relative to contracted controls that had received 200 micrograms/ml ascorbate once. The increase was nearly an order of magnitude over that of controls and was coordinate with a comparable increase in hyaluronate and sulfated glycosaminoglycan (GAG) production as shown by TCA-precipitable glucosamine in the intercellular matrix of the DE. Both the LSE and the Living Dermal Equivalent (LDE) exhibit complex responses to UV radiation and to various chemicals that are greatly different from responses given by monolayered cells.(ABSTRACT TRUNCATED AT 250 WORDS)