Visualization of the polarity of isolated titin molecules: a single globular head on a long thin rod as the M band anchoring domain?

TII, the extractable form of titin, was purified from myofibrils and separated by high resolution gel permeation chromatography into two fractions (TIIA and TIIB). Novel specimen orientation methods used before metal shadowing and EM result in striking pictures of the two forms. Molecules layered on mica become uniformly oriented when subjected to centrifugation. TIIB comprises a very homogeneous fraction. All molecules reveal a single globular head at one end on a long and very thin rod of uniform diameter. The lengths of the rods have a very narrow distribution (900 +/- 50 nm). TIIA molecules seem lateral oligomers of TIIB, attached to each other via the head regions. While dimers are the predominant species, trimers and some higher oligomers can also be discerned. Mild proteolysis destroys the heads and converts TIIA and TIIB into TIIB-like rods. Similar molecules also result from titin purified from myofibrils by certain established purification schemes. Headless titin molecules show in gel electrophoresis only the TII band, while head bearing molecules give rise to two additional polypeptides at 165 and 190 kD. Immunoelectron microscopy of myofibrils identifies both titin-associated proteins as M band constituents. We speculate that in the polar images of TII the globular head region corresponds to the M band end of the titin molecules. This hypothesis is supported by immunoelectron micrographs of TIIB molecules using titin antibodies of known epitope location in the half sarcomere. This proposal complements our previous immunoelectron microscopic data on myofibrils. They showed that epitopes present only on the nonextractable TI species locate to the Z line and its immediately adjacent region (Fürst, D. O., M. Osborn, R. Nave, and K. Weber. 1988. J. Cell Biol. 106:1563-1572). Thus, the two distinct ends of the titin molecule attach to Z and M band material respectively.     

Expression of class I histocompatibility antigens in neuroectodermal tumors is independent of the expression of a transfected neuroblastoma myc gene

We have evaluated the relationship between the neuronal myc gene (NMYC) and class I major histocompatibility complex (MHC) expression in human neuroblastoma (NB) tumor cell lines. Class I MHC surface Ag expression in NB cell lines varied from nearly undetectable to levels nearly as high as in a lymphoblastoid cell line. Class I MHC mRNA levels in NMYC-amplified NB cell lines were lower than levels observed in single copy NMYC NB cell lines. However, considerable variation in class I MHC surface Ag and mRNA expression was evident in NMYC-amplified cell lines. To determine directly whether NMYC might modulate class I MHC expression in NB, we transfected a plasmid containing a recombinant NMYC gene into two tumor cell lines derived from a NB and a related neuroepithelioma tumor. Constitutive overexpression of the recombinant NMYC gene produced no consistent change in class I MHC surface Ag or mRNA levels. To determine whether class I MHC expression might be developmentally regulated in adrenal medullary cells, the precursor cells of adrenal NB tumors, beta 2-microglobulin expression was measured in fetal and adult adrenal glands. beta 2-Microglobulin expression was not evident in the neuroblasts of a 24-wk-old fetal adrenal gland, whereas beta 2-microglobulin expression was present in the adult adrenal medulla. These data suggest that variation in class I MHC expression among NB cells may reflect the developmental stage at which neuroblasts were arrested during tumorigenesis.     

Maturation in Larch : II. Effects of Age on Photosynthesis and Gene Expression in Developing Foliage

The effect of maturation on the morphological and photosynthetic characteristics, as well as the expression of two genes involved in photosynthesis in the developing, current year foliage of Eastern larch (Larix laricina [Du Roi]) is described. These effects were observed on foliage during the third growing season after grafting of scions from trees of different ages onto 2 year old rootstock. Specific leaf weight (gram dry weight per square meter), leaf cross-sectional area (per square millimeter), and chlorophyll content (milligram per gram dry weight) all increase with increasing age in long shoot foliage from both indoor- and outdoor-grown trees. Net photosynthesis (NPS) (mole of CO₂ per square millimeter per second) increases with age on indoor- but not outdoor-grown trees. NPS also increases with increased chlorophyll content, but outdoor-grown scions of all ages had higher chlorophyll content, and chlorophyll does not appear to be limiting for NPS outdoors. To extend these studies of maturation-related differences in foliar morphology and physiology to the molecular genetic level, sequences were cloned from the cab and rbsS gene families of larch. Both cab and rbcS gene families are expressed in foliage but not in roots, and they are expressed in light-grown seedlings of larch but only at very low levels in dark-grown seedlings (~2% of light-grown seedlings). Steady-state cab mRNA levels are relatively higher (~40%) in newly expanding short shoot foliage from juvenile plants compared to mature plants. Unlike cab, the expression of the rbcS gene family did not seem to vary with age. These data show that the maturation-related changes in morphological and physiological phenotypes are associated with changes in gene expression. No causal relationship has been established, however. Indeed, we conclude that the faster growth of juvenile scions reported previously (MS Greenwood, CA Hopper, KW Hutchison [1989] Plant Physiol 90: 406-412) is not due to increased NPS or cab expression. Long shoot foliage is the dominant foliar type on young trees and its lower specific leaf weight will permit production of more photosynthetic surface area per unit of leaf biomass.     

Heavy ion effects on yeast: inhibition of ribosomal RNA synthesis

Diploid wild-type yeast cells were exposed to beams of heavy ions covering a wide range of linear energy transfer (LET) (43-13,700 keV/microns). Synthesis of ribosomal RNA (rRNA) was assessed as a functional measure of damage produced by particle radiation. An exponential decrease of relative rRNA synthesis with particle fluence was demonstrated in all cases. The inactivation cross sections derived were found to increase with LET over the entire range of LET studied. The corresponding values for relative biological effectiveness were slightly less than unity. Maximum cross sections measured were close to 1 micron 2, implying that some larger structure within the yeast nucleus (e.g., the nucleolus) might represent the target for an impairment of synthetic activity by very heavy ions rather than the genes coding for rRNA. Where tested, an oxygen effect for rRNA synthesis could not be demonstrated.     

Relative efficacies of 1,4-diazepines on GABA-stimulated chloride influx in rat brain vesicles

The effects of 1,4-diazepines with two annelated heterocycles [brotizolam (WE 941), ciclotizolam (WE 973) and WE 1008] on gamma-aminobutyric acid (GABA)-stimulated chloride influx into rat brain membrane vesicles were examined. Brotizolam enhanced GABA (30 microM)-stimulated 36Cl- influx (146.1% of control), while ciclotizolam and WE 1008 showed only a small enhancement (119.3% and 119.1%, respectively) of GABA-stimulated 36Cl- uptake. Brotizolam resulted in a left shift of the GABA dose response curve at lower concentrations of GABA (10 microM), while at higher concentrations of GABA (1 mM), brotizolam caused a reduction of the maximal response. The enhancement of GABA-stimulated 36Cl- uptake by brotizolam (0.1 microM) was antagonized by Ro 15-1788. At higher concentration of GABA (300 microM), brotizolam inhibited GABA-stimulated 36Cl- uptake in a dose dependent manner and Ro15-1788 failed to antagonize this effect. These results suggest that 1) brotizolam produces an enhancement of GABA (30 microM)-stimulated chloride influx through the benzodiazepine receptor. 2) brotizolam inhibition of GABA (300 microM)-stimulated chloride influx involves an additional mechanism, and 3) the sedative-hypnotic action of brotizolam may be related to its high efficacy at the benzodiazepine/GABA-gated chloride channel.     

Profilin-actin complexes directly elongate actin filaments at the barbed end.

We demonstrate that the profilin-G-actin complex can elongate actin filaments directly at the barbed end but cannot bind to the pointed end. During elongation, the profilin-actin complex binds to the barbed filament end, whereupon profilin is released, leaving the actin molecule behind. This was first proposed by Tilney [Tilney, L. G., et al. (1983) J. Cell Biol. 97, 112-124] and demonstrated by Pollard and Cooper [(1984) Biochemistry 23, 6631-6641] by electron microscopy. We show that a model without any outside energy supply, in contrast to the mechanism proposed by Pollard and Cooper, can be fitted to our and their [Kaiser et al. (1986) J. Cell Biol. 102, 221-226] findings. Input of outside energy is necessary only if profilin-mediated elongation continues after free G-actin has been lowered to or below the critical concentration observed at the barbed end in the absence of profilin.     

In vitro DNA methylation inhibits gene expression in transgenic tobacco.

A hemimethylated chimeric gene, containing the cauliflower mosaic virus 35S promoter, the beta-glucuronidase coding region and the polyadenylation signal of nopaline synthase, was introduced into tobacco protoplasts by polyethylene glycol mediated transfection. Hemimethylation led to complete inhibition of transient gene expression. In regenerated transgenic plants the integrated gene was constitutively hypermethylated at the sequences CpG and CpNpG and this was correlated with an inactivation of beta-glucuronidase in 12 out of 18 analyzed plant lines whereas two showed slight and four strong activity. From 10 control lines transformed with nonmethylated DNA, only two were inactive; three showed slight and five strong activity. 5-aza-cytidine treatment of plant tissue from 'hypermethylated' lines led to induction of beta-glucuronidase in most cases. Shoots regenerated from azaC treated calli revealed stable enzyme restoration and demethylation of the integrated transgene.     

Concentration dependence of the subunit association of oligomers and viruses and the modification of the latter by urea binding.

A theoretical model is presented that accounts for the facilitation of the pressure dissociation of R17 phage, and for the partial restoration of the concentration dependence of the dissociation, by the presence of subdenaturing concentrations of urea. As an indifferent osmolyte urea should promote the stability of the protein aggregates under pressure, and the decrease in pressure stability with urea concentration demonstrates that such indirect solvent effects are not significant for this case, and that the progressive destabilization is the result of direct protein-urea interactions. By acting as a "homogenizer" of the properties of the phage particles, urea addition converts the pressure-induced deterministic dissociation of the phage into a limited stochastic equilibrium. The model establishes the origin of the uniform progression from the stochastic equilibrium of dimers, to the temperature-dependent and partially concentration-dependent association of tetramers, to the fully deterministic equilibrium observed in many multimers and in the virus capsids.     

Evolutionary plasticity in prokaryotes: A panglossian view

Enzyme directed genetic mechanisms causing random DNA sequence alterations are ubiquitous in both eukaryotes and prokaryotes. A number of molecular geneticist have invoked adaptation through natural selection to account for this fact, however, alternative explanations have also flourished. The population geneticist G.C. Williams has dismissed the possibility of selection for mutator activity on a priori grounds. In this paper, I attempt a refutation of Williams' argument. In addition, I discuss some conceptual problems related to recent claims made by microbiologists on the adaptiveness of molecular variety generators in the evolution of prokaryotes. A distinction is proposed between selection for mutations caused by a mutator activity and selection for the mutator activity proper. The latter requires a concept of fitness different from the one commonly used in microbiology.