Multiple pathways for telomere tethering: functional implications of subnuclear position for heterochromatin formation

Technical advances in the imaging of GFP derivatives in living cells have improved our ability to determine the position and dynamics of specific chromatin loci. This approach, combined with genetics and functional assays, has shed new light on how nuclear compartments facilitate gene repression in yeast.     

Monomeric G-protein-coupled receptor as a functional unit

Rhodopsin, the first purified G-protein-coupled receptor (GPCR), was characterized as a functional monomer 30 year ago, but dimerization of GPCRs recently became the new paradigm of signal transduction. It has even been claimed, on the basis of recent biophysical and biochemical studies, that this new concept could be extended to higher-order oligomerization. Here this view is challenged. The new studies of rhodopsin and other simple (class 1a) GPCRs solubilized in detergent are re-assessed and are compared to the earlier classical studies of rhodopsin and other membrane proteins solubilized in detergent. The new studies are found to strengthen rather than invalidate the conclusions of the early ones and to support a monomeric model for rhodopsin and other class 1a GPCRs. A molecular model is proposed for the functional coupling of a rhodopsin monomeric unit with a G-protein heterotrimer. This model should be valid even for GPCRs that exist as structural dimers.     

RAD50 and NBS1 form a stable complex functional in DNA binding and tethering

The RAD50/MRE11/NBS1 protein complex (RMN) plays an essential role during the early steps of DNA double-strand break (DSB) repair by homologous recombination. Previous data suggest that one important role for RMN in DSB repair is to provide a link between DNA ends. The striking architecture of the complex, a globular domain from which two extended coiled coils protrude, is essential for this function. Due to its DNA-binding activity, ability to form dimers and interact with both RAD50 and NBS1, MRE11 is considered to be crucial for formation and function of RMN. Here, we show the successful expression and purification of a stable complex containing only RAD50 and NBS1 (RN). The characteristic architecture of the complex was not affected by absence of MRE11. Although MRE11 is a DNA-binding protein it was not required for DNA binding per se or DNA-tethering activity of the complex. The stoichiometry of NBS1 in RMN and RN complexes was estimated by SFM-based volume analysis. These data show that in vitro, R, M and N form a variety of stable complexes with variable subunit composition and stoichiometry, which may be physiologically relevant in different aspects of RMN function.     

Repetitive region of calpastatin is a functional unit of the proteinase inhibitor

A cDNA portion coding for one of the repetitive regions of pig heart calpastatin (107 kDa) was subcloned into E. coli plasmid pUC119 to express the portion of the proteinase inhibitor gene in bacteria. The expressed protein was a chimaeric protein whose calpastatin segment (130 amino acid residues) was fused with an amino-terminus portion (7 amino acid residues) of beta-galactosidase. The chimaeric protein could inhibit proteolytic activity of calpain (Ca2+-dependent cysteine proteinase), and maintained properties of the authentic calpastatin concerning inhibition specificity and heat stability. These findings led us to conclude that the repetitive region is a functional unit of the proteinase inhibitor.     

Lipoprotein lipase: size of the functional unit determined by radiation inactivation

Radiation inactivation was used to determine the functional molecular weight of lipoprotein lipase (LPL) in rat heart and adipose tissues. This technique reveals the size of the smallest unit required to carry out the enzyme function. Supernatant fractions of the tissue homogenates were exposed to high energy electrons at -135 degrees C. LPL activity showed a simple exponential decay in all samples tested. Because changes in nutritional state shift the distribution of LPL between the capillary endothelial and parenchymal cells within heart and adipose tissues, fasted and refed rats were used for the radiation studies. The functional molecular weight was calculated to be 127,000 +/- 15,000 (mean +/- SD) daltons for heart and adipose. Thus, the smallest unit required for enzyme function was the same in both of these tissues and did not vary with nutritional state. The data suggest that, compared with LPL monomer sizes reported in the range 55,000 to 72,000, this active unit constitutes a dimer.     

Vesicle tethering: TRAPPing transport carriers

Transport vesicles must dock with the appropriate acceptor membrane to ensure faithful protein delivery. Recent work identifies some of the molecular mechanisms that contribute to the specificity of this reaction.     

Influence of ligament stiffness on the mechanical behavior of a functional spinal unit

Data on the stiffnesses of spinal ligaments are required for analytical studies on the mechanical behavior of spinal segments. Values obtained experimentally vary widely in the literature. A finite element model of an L3/L4 functional spinal unit was used to determine the influence of ligament stiffness on intersegmental rotation and forces in the ligaments. The lowest values for ligament stiffness selected from the literature were used in one set of calculations, and the highest values were simulated in a second set. The nonlinear model was loaded with pure moments of 7.5 and 15 Nm in the three main anatomical planes. The mechanical behavior of the functional spinal unit was strongly influenced by ligament stiffness. In some cases, a ligament with low stiffness does not carry any load, while the same ligament with high stiffness has to carry a high load. This indicates that finite element models of spinal segments have to be validated and that a realistic quantitative prediction of ligament forces is extremely difficult.     

The functional unit of the arom conjugate in Neurospora

The functional unit of arom polyenzyme conjugate of Neurospora crassa was determined by analysis of radiation inactivation of each of the five activities in the conjugate. The functional targets for all five enzymes were in close agreement with the value of 300,000 obtained by conventional hydrodynamic procedure for the native dimeric structure. These data indicate that at least 95% of the functional enzyme system in crude extracts exists in a dimeric form and that both polypeptide chains of the homodimer are required for full activity of each of the five enzymes.     

Lamarckism and epigenetic inheritance: a clarification

Since the 1990s, the terms “Lamarckism” and “Lamarckian” have seen a significant resurgence in biological publications. The discovery of new molecular mechanisms (DNA methylation, histone modifications, RNA interference, etc.) have been interpreted as evidence supporting the reality and efficiency of the inheritance of acquired characters, and thus the revival of Lamarckism. The present paper aims at giving a critical evaluation of such interpretations. I argue that two types of arguments allow to draw a clear distinction between the genuine Lamarckian concept of inheritance of acquired characters and transgenerational epigenetic inheritance. The first concerns the explanandum of the processes under consideration: molecular mechanisms of transgenerational epigenetic inheritance are understood as evolved products of natural selection. This means that the kind of inheritance of acquired characters they might be responsible for is an obligatory emergent feature of evolution, whereas traditional Lamarckisms conceived the inheritance of acquired characters as a property inherent in living matter itself. The second argument concerns the explanans of the inheritance of acquired characters: in light of current knowledge, epigenetic mechanisms are not able to drive adaptive evolution by themselves. Emergent Lamarckian phenomena would be possible if and only if individual epigenetic variation allowed the inheritance of acquired characters to be a factor of unlimited change. This implies specific requirements for epigenetic variation, which I explicitly define and expand upon. I then show that given current knowledge, these requirements are not empirically grounded.     

Functional gene expression domains: defining the functional unit of eukaryotic gene regulation

The term functional domain is often used to describe the region containing the cis acting sequences that regulate a gene locus. "Strong" domain models propose that the domain is a spatially isolated entity consisting of a region of extended accessible chromatin bordered by insulators that have evolved to act as functional boundaries. However, the observation that independently regulated loci can overlap partially or completely raises questions about functional requirements for physically isolated domain structures. An alternative model, the "weak" domain model, proposes that domain structure is determined by the distribution of binding sites for positively acting factors, without a requirement for functional boundaries. The domain would effectively be the region that contains these factor-binding sites. Specificity of promoter-enhancer interactions would play a major role in maintaining the functional autonomy of adjacent genes. Sequences that interfere with these interactions (frequently characterised as insulators) would be selected against if they occurred within the domain but not at the edges, or in the interdomain regions. As a result, insulators would often be found near the borders of domains without necessarily being selected to act as boundaries.