Paul R. Mueller

Myt1: A Membrane-Associated Inhibitory Kinase That Phosphorylates Cdc2 on Both Threonine-14 and Tyrosine-15

Cdc2 is the cyclin-dependent kinase that controls entry of cells into mitosis. Phospho-rylation of Cdc2 on threonine-14 and tyrosine-15 inhibits the activity of the enzyme and prevents premature initiation of mitosis. Although Wee1 has been identified as the kinase that phosphorylates tyrosine-15 in various organisms, the threonine-14-specific kinase has not been isolated. A complementary DNA was cloned from Xenopus that encodes Myt1, a member of the Wee1 family that was discovered to phosphorylate Cdc2 efficiently on both threonine-14 and tyrosine-15. Myt1 is a membrane-associated protein that contains a putative transmembrane segment. Immunodepletion studies suggested that Myt1 is the predominant threonine-14-specific kinase in Xenopus egg extracts. Myt1 activity is highly regulated during the cell cycle, suggesting that this relative of Wee1 plays a role in mitotic control.

Resting and action potentials in experimental bimolecular lipid membranes

Abstract Action potentials are constructed step by step in bimolecular lipid membranes by adjusting the membrane composition, ionic gradients, pH, temperature and the concentration of two proteinaceous adsorbates: an excitability inducing material (EIM) of mol. wt less than 10 5 and protamine sulfate. They show most bioelectric kinetic phenomena and generally conform to the Hodgkin and Huxley theory for action potentials in nerve. The evidence indicates that the system consists of two ion selective channel types. One, produced by EIM, develops a cationic e.m.f.; the other, resulting from a complex between EIM and protamine, develops an anionic e.m.f. Both contain a double gating mechanism showing two negative resistances which are controlled by the voltage and by chemical factors including membrane lipid composition, ionic strength, pH, some alkaloids, acridine and phenothiazine derivatives and divalent ions. The action potentials result from the interplay of e.m.f.s and resistances of the two channel populations each acting as a parallel battery and a voltage dependent variable resistive load on the other coupled via the membrane potential. Some possible molecular mechanisms responsible for the conductance changes are discussed.

Translocators in Bimolecular Lipid Membranes: Their Role in Dissipative and Conservative Bioenergy Transductions

Publisher Summary This chapter explains translocators in bimolecular lipid membranes: their role in dissipative and conservative bioenergy transductions. The theory is consistent with and relies heavily on findings in the ancillary fields of monolayer and surface chemistry. An ionic translocator mechanism is energized chemically to permit the synthesis of active transport and other bioenergy transductions in experimental lipid membranes. In an ideal situation, both the surface energies of the torus and of the bilayer are zero and the bilayer are torn or cut without retracting into the torus or forming a drop. The geometry and framing of the planar bilayer and the presence of special solvents create additional energy terms, which are absent in the spherical bilayers. When some lipid membranes are mechanically broken, they persist as sheets that do not contract into a drop. The extremely low ionic conductance of the bilayer must be attributed to the hydrocarbon region. Translocators show different degrees of functional complexity and are either simple or complex and show selectivity or variable dissociation for the translocated species and are also regulatable, that is, gated either by the translocated entity or by other agents. The energy conversions are linked to a central ion translocating system, which is capable of transporting ions against an electrochemical gradient

Induced excitability in reconstituted cell membrane structure

Abstract When bimolecular lipid membranes adsorb appropriate, as yet unidentified, molecules obtained from various biological sources their resistance falls from108 ω cm2 to103–105 ω cm2 and they then become “active” or “electrically excitable” in the sense that their resistance changes reversibly and regeneratively between two definite values in response to suprathreshold applied voltages. The detailed kinetics of these resistance changes are similar to the “action potential” of frog nerve in 0·1M KCI and are identical to those found in the marine alga Valonia and electronic semiconductor tunnel diodes. In this paper, the kinetic and steady state aspects of these resistance changes are presented, and a theory is developed which accounts for the observed phenomena. This theory is compared with the theory of tunnel diodes.

Ligation‐Mediated PCR for Genomic Sequencing and Footprinting

This unit describes how PCR can be used to exponentially amplify segments of DNA located between two specified primer hybridization sites. A single-sided PCR method is used that initially requires specification of only one primer hybridization site; the second is defined by the ligation-based addition of a unique DNA linker. This linker, together with the flanking gene-specific primer, allows exponential amplification of any fragment of DNA. Because a defined, discrete-length sequence is added to every fragment, complex populations of DNA such as sequence ladders can be amplified intact with retention of single-base resolution. The ligation-based protocol was specifically designed for genomic footprinting and direct sequencing reactions, and is described in this context; it can, however, be used for other applications.

Reconstitution of acetylcholine receptor from Torpedo californica with highly purified phospholipids: Effect of α-tocopherol, phylloquinone, and other terpenoid quinones

Abstract Acetylcholine receptor from Torpedo californica can be incorporated by the cholate dialysis procedure into liposomes prepared with crude soybean phospholipids (asolectin). Vesicles reconstituted with asolectin depleted of neutral lipids or with a mixture of pure phospholipids, are less active in catalyzing carbamylcholine-sensitive Na+ flux. Inclusion of α-tocopherol or certain quinones such as coenzyme Q10 or vitamin K1 during reconstitution yields vesicles with carbamylcholine-sensitive Na+ flux which, under optimal conditions, was considerably higher than that observed with vesicles reconstituted with crude phospholipid mixtures.

Ligation-mediated PCR: Applications to genomic footprinting

A major requirement of the conventional polymerase chain reaction (PCR) is that sequences at both ends of the region to be amplified be known. Ligation-mediated PCR bypasses this by requiring knowledge of only one end initially and then adding the second end by ligation of a unique DNA linker. This method was originally developed for studying in vivo protein:DNA interactions at single-copy genes in complex genomes, but it is applicable to other PCR problems in which only one end of the region to be amplified is known. For example, it has been used for genomic sequencing, in vivo methylation analysis, and gene isolation. For each of these applications, the basic method of adding the second defined end is the same. Of the various single-sided PCR strategies, ligation-mediated PCR is uniquely suited to the amplification of a genomic sequence ladder because it preserves the single base resolution present in the starting material. Here ligation-mediated PCR is discussed in the context of genomic sequencing, with specific reference to in vivo footprinting applications.