A composite transposon 3'' to the cow fetal globin gene binds a sequence specific factor.

Two unusual sequence organizations were found within the beta-globin locus of the cow. Each was a composite, consisting of closely linked Alu-type repeats with a short stretch of genomic non-repetitive sequence, called a lagan, sandwiched between. One lagan was found 3' to the fetal globin gene, while the second lay between the adult globin gene and a globin pseudogene. Southern blot analysis indicated that both lagans appeared twice within the cow haploid genome, with the second copies lying outside the cow beta-globin locus. One of these non-globin locus homologues was cloned and subjected to sequence analysis. Comparison of the DNA sequence data showed that the lagan-Alu composite was transposed as a unit. The lagan 3' to the cow fetal globin gene contains the recognition site for a sequence specific DNA binding factor. This factor was present in extracts from fetal, but not from adult cow tissues.     

Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

Small conductance Ca²⁺-sensitive potassium (SK2) channels are voltage-independent, Ca²⁺-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca²⁺ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca²⁺ and Ca²⁺-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca²⁺ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.     

Mechanistic aspects of promoter binding and chain initiation by RNA polymerase

Summary The physicochemical properties of the interactions of RNA polymerase (RPase) with promoter and nonspecific DNA sequences have been investigated. These show that nonspecific binding is principally an ionic interaction and that promoter binding is more complex, involving nonionic interactions. Nonspecific binding has been shown to be very important in the promoter search, and one-dimensional diffusion can account for the rate at which RPase finds the promoter. Significant differences have been reported in the binding process for various promoters and in the effects of regulatory proteins. Further investigation of these differences will lead to a better understanding of the selectivity and regulation of the initiation process.The pathways of the initiation process have been outlined, by recent studies and considerable progress has been made in determining the rates of interconversion of the intermediate states. A number of questions remain about the detail of initiation and the effects of various parameters on the reactions. Of particular importance is the identification of the point at which the enzyme becomes truly processive. In addition, the step which is rate limiting has not been identified in either the productive or nonproductive process. The mechanistic features of the steps after bond formation are just beginning to yield to investigation.Use of substrate analogs with RPase has led to a picture of the polymerization site according to the ability of the enzyme to incorporate analogs. Base specificity appears to be determined primarily by interaction with the template rather than the enzyme, but the ribose moiety must interact with the site quite specifically. The orientation of the phosphate residues has been determined by NMR, which has also proved to be a valuable probe of the initiation site. At this site base specificity is resident in the enzyme and expressed through the interaction of the base and intrinsic metal, as shown by studies with the Cobalt substituted enzyme. In both initiation and polymerization, the reaction has been shown to proceed by inversion of configuration. Techniques similar to those used for initiation will probably be applied to the polymerization reaction as well, which has not recently received as much attention with respect to mechanism. Functional phenomena such as pausing make the polymerization process particularly promising for producing insight into RPase reactions.     

Pinocytic uptake and intracellular degradation of N-(2-hydroxypropyl)methacrylamide copolymers a potential drug delivery system

Synthetic ¹²⁵I-labelled N-(2-hydroxypropyl)methacrylamide copolymers containing four different, potentially degradable peptidyl side chains were incubated with rat visceral yolk sacs cultured in vitro. All copolymers were captured by fluid-phase pinocytosis and three of the side chains were susceptible to lysosomal hydrolysis, resulting in release of [¹²⁵I]iodotyrosine back into the culture medium. Uptake and degradation was completely inhibited by 2,4-dinitrophenol. The thiol-proteinase inhibitor leupeptin did not affect the rate of pinocytosis, but caused different degrees of inhibition of hydrolysis depending on side chain composition.