The eye as a replicating and diverging, modular developmental unit

Sparked by new discoveries in developmental genetics, few topics have generated as much debate as eye evolution. This is somewhat surprising because the central controversy is not unique to eyes, but is a general theme of developmental genetics: evolutionarily conserved genes are deployed during the development of highly divergent morphological features. In the case of eyes, this paradox has engendered opposing camps entrenched in what has been termed a ‘scientific war’. One camp highlights conserved genetic features, concluding that eyes stem from an ancestral prototype. The opposing camp emphasizes variation, arguing that some eyes must have recruited the same genes after separate morphological origins. Here, I blur the line between these camps and suggest that eyes have often evolved by replication, perhaps through the ectopic expression of a conserved, modular regulatory cascade to produce serially homologous structures that often diverged during evolution. Therefore, morphologically diverse eyes could stem from a single ancestral prototype, yet also result from multiple morphological origins.     

Single strand-containing replicating molecules of circular mitochondrial DNA

Mitochondrial DNAs (mtDNAs) from Chang rat solid hepatomas and Novikoff rat ascites hepatomas were examined in the electron microscope after preparation by the aqueous and by the formamide protein monolayer techniques. MtDNAs from both tumors were found to include double-forked circular molecules with a form and size suggesting they were replicative intermediates. These molecules were of two classes. In molecules of one class, all three segments were apparently totally double stranded. Molecules of the second class were distinguished by the fact that one of the segments spanning the region between the forks in which replication had occurred (the daughter segments) was either totally single stranded, or contained a single-stranded region associated with one of the forks. Daughter segments of both totally double-stranded and single strand-containing replicating molecules varied in length from about 3 to about 80% of the circular contour length of the molecule. Similar classes of replicating molecules were found in mtDNA from regenerating rat liver and chick embryos, indicating them to be normal intermediates in the replication of mtDNA All of the mtDNAs examined included partially single-stranded simple (nonforked) circular molecules. A possible scheme for the replication of mtDNA is presented, based on the different molecular forms observed     

Parrots as key multilinkers in ecosystem structure and functioning

Mutually enhancing organisms can become reciprocal determinants of their distribution, abundance, and demography and thus influence ecosystem structure and dynamics. In addition to the prevailing view of parrots (Psittaciformes) as plant antagonists, we assessed whether they can act as plant mutualists in the dry tropical forest of the Bolivian inter‐Andean valleys, an ecosystem particularly poor in vertebrate frugivores other than parrots (nine species). We hypothesised that if interactions between parrots and their food plants evolved as primarily or facultatively mutualistic, selection should have acted to maximize the strength of their interactions by increasing the amount and variety of resources and services involved in particular pairwise and community–wide interaction contexts. Food plants showed different growth habits across a wide phylogenetic spectrum, implying that parrots behave as super‐generalists exploiting resources differing in phenology, type, biomass, and rewards from a high diversity of plants (113 species from 38 families). Through their feeding activities, parrots provided multiple services acting as genetic linkers, seed facilitators for secondary dispersers, and plant protectors, and therefore can be considered key mutualists with a pervasive impact on plant assemblages. The number of complementary and redundant mutualistic functions provided by parrots to each plant species was positively related to the number of different kinds of food extracted from them. These mutually enhancing interactions were reflected in species‐level properties (e.g., biomass or dominance) of both partners, as a likely consequence of the temporal convergence of eco‐(co)evolutionary dynamics shaping the ongoing structure and organization of the ecosystem. A full assessment of the, thus far largely overlooked, parrot–plant mutualisms and other ecological linkages could change the current perception of the role of parrots in the structure, organization, and functioning of ecosystems.     

A 3,4-epoxypiperidine structure as a novel and simple DNA-cleavage unit

Based on the 4-hydroxy-1-azabicyclo[3.1.0]hexane structure of azinomycin, a 3,4-epoxypiperidine structure was designed as a novel and simple alkylating molecular unit, and some 3,4-epoxypiperidine derivatives were found to show DNA-cleavage activity, the structural requirements for which were revealed.     

The structure of replicating kinetoplast DNA networks

Kinetoplast DNA (kDNA), the mitochondrial DNA of Crithidia fasciculata and related trypanosomatids, is a network containing approximately 5,000 covalently closed minicircles which are topologically interlocked. kDNA synthesis involves release of covalently closed minicircles from the network, and, after replication of the free minicircles, reattachment of the nicked or gapped progeny minicircles to the network periphery. We have investigated this process by electron microscopy of networks at different stages of replication. The distribution of nicked and closed minicircles is easily detectable either by autoradiography of networks radiolabeled at endogenous nicks by nick translation or by twisting the covalently closed minicircles with intercalating dye. The location of newly synthesized minicircles within the network is determined by autoradiography of network is determined by autoradiography of networks labeled in vivo with a pulse of [3H]thymidine. These studies have clarified structural changes in the network during replication, the timing of repair of nicked minicircles after replication, and the mechanism of division of the network.     

Nodule structure and functioning in Cicer arietinum as affected by nitrate

Chickpea (Cicer arietinum L.) cv. C-235 inoculated with Rhizobium sp. (Cicer) strain cv4Az was raised in sand culture under natural conditions with nitrogen-free nutrient solution. 45-d-old plants were treated with 20 and 50 mM KNO3 and sampling made 2 and 6 d after treatment. KNO3 application induced premature nodule senescence. Light microscopic investigations showed that KNO3 treatments resulted in structural degradation of the central bacteroidal tissue. The mass of green nodules increased by 35 % under these treatments. This was accompanied by a rapid decline in leghemoglobin (Lb) content of the nodules being 51 - 67 % lower than in control. The total soluble nodule proteins showed relatively minor changes under KNO3 treatments thus suggesting preferential degradation of Lb. These changes were associated with a rapid decline in N2-fixing activity. However, the decline in total soluble sugars was relatively minor as compared to acetylene reducing activity, thus indicating that sugar deprivation is not the cause of decreased nitrogen fixation ability. Glutathione reductase and ascorbate peroxidase activity showed a 10 - 20 % decrease in comparison with the control. Accumulation of H2O2 and structural degradation of the nodular tissue are considered to be the factors leading to nodule senescence under nitrate treatments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Human mitochondria and mitochondrial genome function as a single dynamic cellular unit

rho 0 HeLa cells entirely lacking mitochondrial DNA (mtDNA) and mitochondrial transfection techniques were used to examine intermitochondrial interactions between mitochondria with and without mtDNA, and also between those with wild-type (wt) and mutant-type mtDNA in living human cells. First, unambiguous evidence was obtained that the DNA-binding dyes ethidium bromide (EtBr) and 4',6-diamidino-2- phenylindole (DAPI) exclusively stained mitochondria containing mtDNA in living human cells. Then, using EtBr or DAPI fluorescence as a probe, mtDNA was shown to spread rapidly to all rho 0 HeLa mitochondria when EtBr- or DAPI-stained HeLa mitochondria were introduced into rho 0 HeLa cells. Moreover, coexisting wt-mtDNA and mutant mtDNA with a large deletion (delta-mtDNA) were shown to mix homogeneously throughout mitochondria, not to remain segregated by use of electron microscopic analysis of cytochrome c oxidase activities of individual mitochondria as a probe to identify mitochondria with predominantly wt- or delta- mtDNA in single cells. This rapid diffusion of mtDNA and the resultant homogeneous distribution of the heteroplasmic wt- and delta-mtDNA molecules throughout mitochondria in a cell suggest that the mitochondria in living human cells have lost their individuality. Thus, the actual number of mitochondria per cell is not of crucial importance, and mitochondria in a cell should be considered as a virtually single dynamic unit.     

The structure of replicating adenovirus 2 DNA molecules

Adenovirus 2 (Ad2)-infected KB cells were exposed to a 2.5 min pulse of ³H-thymidine at 19 hr after infection. The labeled DNA molecules were separated from cell DNA and mature Ad2 DNA by sucrose gradient sedimentation and CsCI equilibrium centrifugation under conditions designed to minimize branch migration and hybridization of single strands. Electron microscopy-of fractions containing radioactivity revealed two basic types of putative replicating molecules: Ad2 length duplex DNA molecules with one or more single-stranded branches (type I) and Ad2 length linear DNA molecules with a single-stranded region extending a variable distance from one end (type II). Length measurements, partial denaturation studies and 3′ terminal labeling experiments were consistent with the following model for Ad2 DNA replication. Initiation of DNA synthesis occurs at or near an end of the Ad2 duplex. Following initiation, a daughter strand is synthesized in the 5′ to 3′ direction, displacing the parental strand with the same polarity. This results in the formation of a branched replicating molecule (type I). Initiations at the right and left molecular ends are approximately equal in frequency, and multiple initiations on the same replicating molecule are common. At any given displacement fork in a type I molecule, only one of the two parental strands is replicated. Two nonexclusive mechanisms are proposed to account for the replication of the other parental strand. In some cases, before completion of a round of displacement synthesis initiated at one end of the Ad2 duplex, a second initiation will occur at the opposite end. In these doubly initiated molecules, both parental strands serve as templates for displacement synthesis. Two type II molecules are generated when the oppositely moving displacement forks meet. Alternatively, displacement synthesis may proceed to the end of the Ad2 duplex, resulting in the formation of a daughter duplex and a parental single strand. Replication of the displaced parental strand is then initiated at or near its 3′ terminus, producing a type II molecule. Daughter strand synthesis proceeds in the 5′ to 3′ direction in type II molecules generated by either mechanism, and completion of synthesis results in the formation of a daughter duplex.     

Age-related changes in the functioning of the mitochondrial potassium-transporting system

Changes in the rate of respiration and functioning of the ATP-dependent potassium channel in liver and heart mitochondria of one-, three-, eight-, and 24-month-old Wistar male rats have been investigated. It was shown that the activity of the channel in the mitochondria of both tissues in 24-month-old animals decreases more than three times, and the content of potassium, 1.5–2 times compared with young one-month-old rats. The changes occur against the background of age-related decrease of energy supply in mitochondria, the respiratory complex-I undergoing the greatest changes upon aging. The decrease of channel activity may be the result of changes in channel sensitivity to modulators and a decrease in the expression of mitochondrial K+-transporting channel-protein with a molecular mass of 5.5 kDa upon aging found in this work. As a result, the functioning of not only the mitoK_ATP but also the whole mitochondrial potassium cycle is impaired.     

Features of the structure of replicating and non-replicating chromatin in chicken erythroblasts.

The digestion by DNAase I of DNA synthesised by isolated chicken erythroblasts was examined in isolated nuclei. It was found that newly synthesised DNA was susceptible to DNAase I but matured to a relatively resistant form with increasing time after replication as observed in mammalian systems. The presence of trypsin in the digestion exposed all of the DNA to DNAase I action. Examination of the digestion products showed that the newly replicated DNA differed little from the more mature form in the structure of the DNA-protein complex but that the difference in susceptibility was probably a result of a differential rate of access of the DNAase to the new and old DNA.     

Autonomously replicating fragments of mitochondrial DNA from Penicillium urticae in Saccharomyces cerevisiae

Eight fragments which cover the whole range of the mitochondrial genome of Penicillium urticae were subcloned into the yeast integration vector YIp5. Transformation of Saccharomyces cerevisiae with the constructed plasmids by the alkali cation method indicated that six plasmids are able to replicate in yeast. Both closed and open circular forms of the plasmids were detected in the DNA extracts from transformants. Distribution of the autonomously replicating sequence in the mitochondrial genome was similar to that in P. chrysogenum except for one small region.     

Two autonomously replicating sequences near oli-1 gene of yeast mitochondrial DNA

We cloned two autonomously replicating sequences from a short segment of mtDNA of an oligomycin-resistant petite yeast, O-111, into a vector pYleu 12 constructed from yeast LEU 2 gene and pBR 322. These plasmids, pYmit 4 and pYmit 1, had frequencies of transformation of yeast as high as that of YEp 13, having a replicator of 2 mu DNA. They were maintained as plasmids in yeast under selective conditions and shuttled from yeast to E. coli. No evidence was obtained that these plasmids were incompatible with the wild-type mitochondrial genome. These sequences were located in intergenic regions.     

A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a very large membrane protein complex with a central function in energy metabolism. Complex I from the aerobic yeast Yarrowia lipolytica comprises 14 central subunits that harbour the bioenergetic core functions and at least 28 accessory subunits. Despite progress in structure determination, the position of individual accessory subunits in the enzyme complex remains largely unknown. Proteomic analysis of subcomplex Iδ revealed that it lacked eleven subunits, including the central subunits ND1 and ND3 forming the interface between the peripheral and the membrane arm in bacterial complex I. This unexpected observation provided insight into the structural organization of the connection between the two major parts of mitochondrial complex I. Combining recent structural information, biochemical evidence on the assignment of individual subunits to the subdomains of complex I and sequence-based predictions for the targeting of subunits to different mitochondrial compartments, we derived a model for the arrangement of the subunits in the membrane arm of mitochondrial complex I.     

Solution structure of the CBM10 cellulose binding module from Pseudomonas xylanase A

Plant cell wall hydrolases generally have a modular structure consisting of a catalytic domain linked to one or more noncatalytic carbohydrate-binding modules (CBMs), whose common function is to attach the enzyme to the polymeric substrate. Xylanase A from Pseudomonas fluorescens subsp. cellulosa (Pf Xyn10A) consists of a family 10 catalytic domain, an N-terminal family IIa cellulose-binding module, and an internal family 10 cellulose-binding module. The structure of the 45-residue family 10 CBM has been determined in solution using NMR. It consists of two antiparallel beta-sheets, one with two strands and one with three, with a short alpha-helix across one face of the three-stranded sheet. There is a high density of aromatic residues on one side of the protein, including three aromatic residues (Tyr8, Trp22, and Trp24), which are exposed and form a flat surface on one face, in a classical polysaccharide-binding arrangement. The fold is closely similar to that of the oligonucleotide/oligosaccharide-binding (OB) fold, but appears to have arisen by convergent evolution, because there is no sequence similarity, and the presumed binding sites are on different faces.