Structural basis for the activity of two muconate cycloisomerase variants toward substituted muconates.

We have refined to 2.3 A resolution two muconate cycloisomerase (MCIase) variant structures, F329I and I54V, that differ from each other and from wild-type in their activity toward cis,cis-muconate (CCM) and substituted CCMs. The working and free R-factors for F329I are 17.4/21.6% and for I54V, 17.6/22.3% with good stereochemistry. Except for the mutated residue, there are no significant changes in structure. To understand the differences in enzymatic properties we docked substituted CCMs and CCM into the active sites of the variants and wild type. The extra space the mutations create appears to account for most of the enzymatic differences. The lack of other structural changes explains why, although structurally equivalent changes occur in chloromuconate cycloisomerase (CMCIase), the changes in themselves do not convert a MCIase into a dehalogenating CMCIase. Reanalysis of the CMCIase structure revealed only one general acid/base, K169. The structural implication is that, in 2-chloro-CCM conversion by CMCIase, the lactone ring of 5-chloromuconolactone rotates before dehalogenation to bring the acidic C4 proton next to K169. Therefore, K169 alone performs both required protonation and deprotonation steps, the first at C5 as in MCIase, and the second, after ring rotation, at C4. This distinguishes CMCIase from alpha/beta barrel isomerases and racemases, which use two different bases.     

A statistical model of the timing mechanism of the erythrocyte life-span

Three statistical models are introduced as explanations of erythrocyte senescence. For the sake of simplicity, we prefer the multievent model, in which one of the parameters represents the number of events (between 7 and 12) needed to signal likely senescence. Proteolysis of a dimeric protein, such as protein 3, accounts for four events in the model, if little of the protein is degraded by senescence. Although each model we propose accommodates the possibility of random destruction, this alternative does not fit well with available experimental data.     

The primary sequence of a crustacean satellite DNA containing a family of repeats

The repeating sequence of isopycnic twin Satellite II of the hermit crab, Pagurus pollicaris, has been determined. More than 95% of the primary sequence of the light strand is composed of (CAG)nCTGCACT. The only detectable variation on the repeating sequence is the number of CAGs included in each repeat which varies from 3-12. The sequence of Satellite II differs markedly from that of its twin, whose light strand is greater than 95% CCTA (Skinner et al., 1974). The absence from Satellite II of repeats where (CAG)n less than or equal to 2 further defines the constraints on a recent model for the origin of satellites (Smith, 1976).     

Bacterial and firefly luciferase genes in transgenic plants: Advantages and disadvantages of a reporter gene

Genes encoding light-emitting luciferase were recently isolated from luminous marine bacteria and fireflies. Expression of luciferase genes in diverse organisms is a unique way for studying gene expression by simple and sensitive measurement of light. Recent advances in application of luciferase reporter genes are reviewed and documented by examples of in vivo visualization of their expression in transgenic plants.     

Allotetraploid hybrids between citrus and seven related genera produced by somatic hybridization

We have developed an efficient protoplast-fusion method to produce somatic hybrid allopolyploid plants that combine Citrus with seven related genera, including four that are sexually incompatible. In this paper we report the creation of 18 new allotetraploid hybrids of Citrus, including ten among sexually incompatible related genera, that may have direct cultivar potential as improved citrus rootstocks. All hybrids were confirmed by cytological and RAPD analyses. If fertile, the attributes of these hybrids may be amenable to further genetic manipulation by breeding at the tetraploid level. Wide somatic hybridization of Citrus via protoplast fusion bypasses biological barriers to the natural allopolyploidization of Citrus, and creates new evolutionary opportunities that would be difficult or impossible to achieve by natural or conventional hybridization.Florida Agricultural Experiment Station Journal Series No. R-04520