Origin of a gene regulatory mechanism in the evolution of echinoderms

A rich diversity of ancient sea urchin lineages survives to the present. These include several advanced orders as well as the cidaroids, which represent the group ancestral to all other sea urchins. Here we show that all advanced groups of sea urchins examined possess in their eggs a class of maternal messenger RNA (mRNA) encoded by the evolutionarily highly conserved alpha-subtype histone genes. The maternal histone mRNAs are unique in their time of accumulation in oogenesis, their localization in the egg nucleus and their delayed timing of translation after fertilization. Cidaroid sea urchins as well as other echinoderm classes, such as starfish and sea cucumbers, possess the genes but do not have maternal alpha-subtype histone mRNAs in their eggs. Thus, although all the echinoderms examined transcribe alpha-subtype histone genes during embryogenesis, the expression of these genes as maternal mRNAs is confined to advanced sea urchins. The fossil record allows us to pinpoint the evolution of this mode of expression of alpha-histone genes to the time of the splitting of advanced sea urchin lineages from the ancestral cidaroids in a radiation which occurred in a relatively brief interval of time approximately 190-200 Myr ago. The origin of a unique gene regulatory mechanism can thus be correlated with a set of macroevolutionary events.     

Tissue-specific activation of a cloned alpha-fetoprotein gene during differentiation of a transfected embryonal carcinoma cell line

To identify cis-acting DNA elements involved in the activation of the alpha-fetoprotein gene during differentiation, modified copies of the gene were introduced into murine F9 embryonal carcinoma cells. The differentiation of the transformants to either parietal or visceral endoderm was accompanied by induction of the exogenous template in a manner qualitatively, but not quantitatively, identical to that of the endogenous alpha-fetoprotein gene.     

A six-armed oligomer isolated from cell surface fibronectin preparations

Fibronectins are adhesive glycoproteins thought to mediate the attachment of cells to various substrates. Plasma fibronectin (PFN) is a dimer comprising subunits of molecular weight 220,000, connected by one or two disulphide bonds. Electron microscopy shows that PFN is a long, flexible strand, 2-3 nm in diameter and 140 nm long. Many cells in tissue culture elaborate an extracellular matrix of insoluble (highly cross-linked by disulphide bonds) fibronectin, and a variable amount of 'cell surface fibronectin' (CSFN) that can be extracted by mild urea treatment. This CSFN, soluble in 1 M urea and at high pH, is a mixture of dimers and disulphide-bonded oligomers. In the present study we have examined the structure of these molecules by electron microscopy. Oligomers were separated from dimers and contaminating proteins by zone sedimentation through glycerol gradients. We report that the CSFN dimers are identical in structure to PFN. In contrast, the oligomers have an elaborate and well defined structure that we call a 'hexabrachion': six arms emanating from a central globular particle. The arms are similar to PFN in being long, thin and flexible, but have several distinctly different features.     

Two types of cholinergic innervation in cortex, one co-localized with vasoactive intestinal polypeptide

The existence of cholinergic neuronal cell bodies in mammalian cerebral cortex was long the subject of much controversy (see ref. 1 for review). Recently, however, a specific cholinergic marker, the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT, E.C.2.3.1.6), was demonstrated by immunohistochemical methods to be present in bipolar neurones in rat cortex. Here we show that at least 80% of these intrinsic cholinergic neurones also contain immunoreactivity for vasoactive intestinal polypeptide (VIP), a neuroactive peptide found to be present in a subpopulation of cortical neurones. On the other hand, we find that the ChAT-positive cells in the basal forebrain, which are another major source of cholinergic innervation of the cortex, contain no detectable VIP-immunoreactivity. In addition, we have observed by both light and electron microscopy that some VIP- and some ChAT-positive structures in cortex are closely associated with blood vessels.     

Iscom, a novel structure for antigenic presentation of membrane proteins from enveloped viruses

We describe here a novel type of immunostimulating complex, called 'iscom', in which virus membrane proteins are presented in a multimeric form. The matrix of the iscom is the glycoside Quil A (Spikoside; Iscotec AB), extracted from the bark of Quillaja saponaria Molina, which forms micelles at the critical micellar concentration of 0.03%. In micelle form, Quil A probably has regions accessible for hydrophobic interaction with the membrane proteins so that it can form complexes with them. Iscoms have been prepared with membrane proteins of para-influenza-3 (PI-3), measles and rabies viruses, and their immunizing potency tested in animals. In these experiments, iscoms prove to be at least 10 times more potent than micelles formed by aggregation of the membrane proteins alone. Iscoms of PI-3 and measles viruses also stimulate the formation of antibody to the fusion (F) protein, which is considered to be poorly immunogenic. No side effects of iscoms or of protein micelles have been observed.     

Gelsolin inhibition of fast axonal transport indicates a requirement for actin microfilaments

The actions of actin-based microfilaments in cell motility suggest a possible role in the mechanism of fast axonal transport, but the pharmacological data evaluating their role in this process are equivocal. Moreover, microfilaments are difficult to preserve and identify in ultrastructural studies, so the organization and function of axonal actin has remained uncertain. We have now evaluated the role of actin microfilaments in intracellular transport of membranous organelles using video-enhanced contrast microscopy and gelsolin to analyse fast axonal transport directly in isolated axoplasm from the squid giant axon. With this preparation it is possible to perfuse axoplasm with large molecules that do not cross the plasmalemma, while controlling cation levels. The 90,000-molecular weight protein gelsolin depolymerizes actin microfilaments in micromolar Ca2+, but not in the absence of Ca2+. Axonal transport of membranous organelles has previously been shown to be unaffected by levels of Ca2+ up to 10 microM. In the presence of EGTA, gelsolin has no effect on the movement of membranous organelles, but in the presence of 10 microM Ca2+ it completely blocks transport of all membranous organelles. No changes in the organization of the axoplasm were detected. These results and results using other probes for actin are consistent with the hypothesis that actin-based microfilaments are involved in the movement of membranous organelles in the axon.     

Direct addition of insulin inhibits a high affinity Ca2+-ATPase in isolated adipocyte plasma membranes.

The mechanism by which insulin regulates cellular metabolism remains unknown although indirect evidence suggests that alterations in intracellular calcium are important. More specifically, it has been proposed that insulin triggers an increase in intracellular calcium which is responsible for the subsequent modification of metabolic activities. The cell maintains a large electrochemical gradient for ionised calcium between the cytoplasm (less than 10(-6) M, as determined for muscle and nerve) and the extracellular environment (less than 10(-3) M). The plasma membrane may, therefore, be important in the regulation of calcium homeostasis, as a slight alteration in the processes maintaining this gradient could result in marked changes in cytoplasmic calcium. One such process is the active extrusion of calcium from the cell by a high affinity calcium-stimulated ATPase (Ca2+-ATPase). Such a mechanism has been well established in red cells and is postulated in nerve, liver and muscle. We have identified a high affinity Ca2+-ATPase in a plasma membrane-enriched subcellular fraction isolated from rat adipocytes which may provide the enzymatic basis for a calcium extrusion pump. We demonstrate here that the Ca2+-ATPase is specifically inhibited by the direct addition of physiological concentrations of insulin to the direct addition of physiological concentrations of insulin to the isolated plasma membranes. This effect suggests that direct regulation of calcium homeostasis may represent an important event in the mechanism of action of insulin.     

Endogenous mammary tumour virus DNA varies among wild mice and segregates during inbreeding.

Proviruses of the mouse mammary tumour virus (MMTV) endogenous to normal mice can be identified by molecular hybridisation and distinguished using restriction endonucleases. Feral mice display marked heterogeneity with respect to the number of copies and the sites of insertion of endogenous MMTV-specific DNA, with occasional mice apparently free of MMTV DNA. Several different MMTV proviruses present in laboratory mice have segregated like stable, independent genetic elements during the inbreeding which followed a cross between Bagg albino and DBA mice 60 years ago. The results favour the hypothesis that endogenous proviruses have been established by multiple, independent infections of germ cells rather than by somatic mutation of ancestral proviruses or of cellular genes.     

Modulation of the two promoters of the galactose operon of Escherichia coli.

The gal operon of Escherichia coli is controlled by two independent promotors--one is activated and the other inhibited by cyclic AMP and cyclic AMP receptor protein. The two promotors are modulated, however, by the same operator locus and receptor protein.