Light-dependent conformational change at rhodopsin's cytoplasmic surface detected by increased susceptibility to proteolysis

Rhodopsin in bovine photoreceptor disk membranes was subjected to limited proteolysis by thermolysin, removing twelve amino acids from rhodopsin's carboxyl terminus. (1) The rate of proteolysis is significantly faster with rhodopsin following exposure to light than with unbleached rhodopsin, provided that the incubation conditions (pH, temperature) favor the formation of metarhodopsin II. (2) If the disk membranes are illuminated under conditions in which metarhodopsin I is the predominant photoproduct (pH 8.5, 0°C), no increase in the rate of proteolysis is observed compared to unilluminated membranes. (3) The light-induced increase in the rate of proteolysis is transient: it slowly decays in the dark to the original rate found for unbleached rhodopsin. The enhanced susceptibility to proteolysis appears to measure a conformational change at rhodopsin's cytoplasmic surface which is first exhibited at the metarhodopsin II stage. This and possibly other light-dependent changes may allow rhodopsin to mediate its signal as a light-receptor protein by binding to and activating certain rod cell enzymes.     

Binding of 4-methylumbelliferyl-galactosides to peanut agglutinin

The binding of 4-methylumbelliferyl-α-D-galactopyranoside, -β-D-galactopyranoside and -D-Galβ(1→3)DGalNac to peanut agglutinin was studied by fluorescence. Peanut agglutinin quenched the fluorescence intensity of 4-methylumbelliferyl-α-D-galactopyranoside but enhanced that of the two 4-methylumbelliferyl-β-galactosides. For α-D-galactopyranoside, the association constants measured at 4 and 25°C were 3.4 × 10³ and 1.7 × 10³ M^?1 respectively, and for D-Galβ(1→3)DGalNac, 1.5 × 10⁵ and 3.3 × 10⁴ M^?1. The binding enthalpies estimated from these values are consistent with the existence of extended sugar binding sites in the peanut agglutinin molecule.     

Mitochondrial DNA evolution in lagomorphs: Origin of systematic heteroplasmy and organization of diversity in European rabbits

Summary A characterization was conducted on mitochondrial DNA (mtDNA) molecules extracted separately from 107 European rabbits (Oryctolagus cuniculus) both wild and domestic, 13 European hares (Lepus capensis), and 1 eastern cottontail (Sylvilagus floridanus). Experimentally this study took into account restriction site polymorphism, overall length variation of the noncoding region, and numbers of repeated sequences. Nucleotide divergences indicate that the mtDNAs from the three species derived from a common ancestor some 6–8 million years (Myr) ago. Every animal appeared heteroplasmic for a set of molecules with various lengths of the noncoding region and variable numbers of repeated sequences that contribute to them. This systematic heteroplasmy, most probably generated by a rate of localized mtDNA rearrangements high enough to counterbalance the cellular segregation of rearranged molecules, is a shared derived character of leporids.The geographic distribution of mtDNA polymorphism among wild rabbit populations over the western European basin shows that two molecular lineages are represented, one in southern Spain, the second over northern Spain, France, and Tunisia. These two lineages derived from a common ancestor some 2 Myr ago. Their present geographical distribution may be correlated to the separation of rabbits into two stocks at the time of Mindel glaciation.Finally the distribution of mtDNA diversity exhibits a mosaic pattern both at inter- and intrapopulation levels.     

Sequences homologous to Tc(s) transposable elements ofCaenorhabditis elegans are widely distributed in the phylum nematoda

Summary To have a better understanding of the evolutionary history of mobile elements within the nematodes, we examined the distribution and the conservation of homologues to transposable elements fromCaenorhabditis elegans (Tc1, Tc2, Tc3, Tc4, Tc5, and FB1) in 19 nematode species belonging to the class Secernentea. Our results show that Tc1 elements display a distribution restricted to the family Rhabditidae with poor conservation. The Tc2 and FB1 homologous elements have the same patchy distribution within the Rhabditidae. They were only found inCaenorhabditis and inTeratorhabditis. The Tc3 element is widely distributed among nematode species. Tc3 homologous elements are present in the majority of the Rhabditidae but also in two genera within the family Panagrolaimidae, and inBursaphelenchus, which belongs to the order Aphelenchida. Tc4 and Tc5 homologues show the most limited distribution of all tested elements, being strictly limited toC. elegans. These data indicate that in some cases, the distribution of transposable elements in the nematode cannot be explained by strict vertical transmission. The distribution of Tc3, Tc4, and Tc5 suggests that horizontal transmission may have occurred between reproductively isolated species during their evolutionary history.     

High precision immunoscanning electron microscopy using Fab fragments coupled to ultra-small colloidal gold.

Ultra-small colloidal gold (less than 1 nm), bound to Fab fragments provides the shortest practical specific marker system to date and can be used in concert with field emission scanning electron microscopes to precisely locate antigenic sites. An "in-lens" FE-SEM equipped with a highly sensitive single crystal YAG-detector for backscattered electrons, as well as the use of advanced specimen preparation techniques based on cryofixation, are among the indispensible prerequisites. A T-even type Escherichia coli bacteriophage, Tu II*-46, was chosen to study properties of the immunogold labeling system. Distinct regions on the tail fibers of this phage were labeled with Fab fragments derived from antibodies against the related phage Tu II*-6. The tail fibers are composed of pairs of homologous proteins, thus offering two identical antigenic sites at the same locus on the tail fibers. Fab fragments can be visualized in the SEM at high accelerating voltage (30 kV) without any additional marker. This permits comparison of the labeling characteristics of unmarked and colloidal gold-marked Fab fragments. Unmarked Fab fragments often bind by pairs (two singlet Fab fragments bound opposed to each other along the axis of the tail fiber). The labeling efficiency of unmarked Fab fragments was greater than that of ultra-small gold-labeled Fab fragments. Binding by pairs was not seen after labeling with ultra-small gold-Fab fragments. The conjugates used in this study exhibited one colloidal gold per Fab fragment.     

Control of the Lysine Biosynthesis Sequence in Corynebacterium glutamicum as Analyzed by Overexpression of the Individual Corresponding Genes

The gene cluster that codes for feedback-resistant aspartate kinase (lysCα and lysCβ) and aspartate semialdehyde dehydrogenase (asd) was cloned from a mutant strain of Corynebacterium glutamicum. Its functional analysis by subcloning, enzyme assays, and type of aspartate kinase regulation enabled the isolation of a fragment for separate expression of the feedback-resistant kinase without aspartate semialdehyde dehydrogenase expression. This was used together with other clones constructed (J. Cremer, L. Eggeling, and H. Sahm, Mol. Gen. Genet. 220:478-480, 1990) to overexpress individually each of the six genes that convert aspartate to lysine. Analysis of lysine formation revealed that overexpression of the feedback-resistant kinase alone suffices to achieve lysine formation (38 mM). Also, sole overexpression of wild-type dihydrodipicolinate synthase resulted in lysine formation but in a lower amount (11 mM). The other four enzymes had no effect on lysine secretion. With a plasmid overexpressing both relevant enzymes together, a further increase in lysine yield was obtained. This shows that of the six enzymes that convert aspartate to lysine the kinase and the synthase are responsible for flow control in the wild-type background and can be useful for construction of lysine-producing strains.     

Stimulation of the anaerobic growth ofSalmonella typhimurium by reduction ofl-carnitine,carnitine derivatives and structure-related trimethylammonium compounds

In view of the development of al-carnitine deficiency, the metabolism ofl-carnitine and structure-related trimethylammonium compounds was studied inSalmonella typhimurium LT₂ by means of thin-layer chromatography (TLC).l-Carnitine, crotonobetaine and acetyl-l-carnitine stimulated the anaerobic growth in a complex medium significantly. The stimulation depended on the formation of -butyrobetaine. The reduction ofl-carnitine proceeded in two steps: (1) Dehydration of thel-carnitine to crotonobetaine, (2) hydrogenation of crotonobetaine to -butyrobetaine. The reduction of crotonobetaine was responsible for the growth stimulation. Terminal electron acceptors of the anaerobic respiration such as nitrate and trimethylamine N-oxide, but not fumarate, suppressed the catabolism ofl-carnitine completely. Glucose fermentation, too, inhibited the reduction ofl-carnitine but optimal growth with a high carnitine catabolism was achieved byd-ribose. The esters of carnitine with medium- and long-chain fatty acids inhibited the growth considerably because of their detergent properties.Abbreviations TLC thin-layer chromatography     

Hybridization of a mouse T-cell lymphocyte line (HB1) with a polyoma virus-transformed mouse fibroblast line

The establishment of permanent T-lymphocyte cell lines by transformation with DNA viruses has not yet been achieved. This paper reports the successful transfer of polyoma virus genome into T-lymphocyte cells by somatic hybridization. A T-lymphocyte clone, HB₁, derived from (DBA/ 2J×AKR) spleen cells, isolated in vitro by cloning in semi-solid agar, was fused with a polyoma (Py) virus-transformed fibroblast C3HPy, clone 1. The authenticity of the hybrid C3H/HB was established by chromosome and histocompatibility antigen studies. This initial population and the various clones retained T-lymphocyte characteristics such as morphological appearance, growth properties (suspension culture) and differentiation antigen (Thy 1–2). The hybrid cell line and the various clones presented all the characteristics of Py transformation. Namely, they carried the Py genome originating from the fibroblastic parent and maintained Py virus tumour-associated antigens (TSTA, TSSA and T antigens). In most respects, this hybrid population resembled the C3HPy/C11 parent and exhibited the same tumorigenicity.