In vitro reconstruction of the Aspergillus (= Emericella) nidulans genome

A physical map of the 31-megabase Aspergillus nidulans genome is reported, in which 94% of 5,134 cosmids are assigned to 49 contiguous segments. The physical map is the result of a two-way ordering process, in which clones and probes were ordered simultaneously on a binary DNA/DNA hybridization matrix. Compression by elimination of redundant clones resulted in a minimal map, which is a chromosome walk. Repetitive DNA is nonrandomly dispersed in the A. nidulans genome, reminiscent of heterochromatic banding patterns of higher eukaryotes. We hypothesize gene clusters may arise by horizontal transfer and spread by transposition to explain the nonrandom pattern of repeats along chromosomes.

Three-dimensional reconstruction of the recombinant type 3 ryanodine receptor and localization of its amino terminus

Recombinant type 3 ryanodine receptor (RyR3) has been purified in quantities sufficient for structural characterization by cryoelectron microscopy and three-dimensional (3D) reconstruction. Two cDNAs were prepared and expressed in HEK293 cells, one encoding the wild-type RyR3 and the other encoding RyR3 containing glutathione S-transferase (GST) fused to its amino terminus (GST-RyR3). RyR3 was purified from detergent-solubilized transfected cells by affinity chromatography using 12.6-kDa FK506-binding protein in the form of a GST fusion as the affinity ligand. Purification of GST-RyR3 was achieved by affinity chromatography by using glutathione-Sepharose. Purified recombinant RyR3 and GST-RyR3 proteins exhibited high-affinity [3H]ryanodine binding that was sensitive to activation by Ca2+ and caffeine and to inhibition by Mg2+. 3D reconstructions of both recombinant RyR3 and GST-RyR3 appeared very similar to that of the native RyR3 purified from bovine diaphragm. Comparison of the 3D reconstructions of RyR3 and GST-RyR3 revealed that the GST domains and, hence, the amino termini of the RyR3 subunits are located in the “clamp” structures that form the corners of the square-shaped cytoplasmic region of homotetrameric RyR3. This study describes the 3D reconstruction of a recombinant ryanodine receptor and it demonstrates the potential of this technology for characterizing functional and structural perturbations introduced by site-directed mutagenesis.

Evolutionary reconstruction of a hairpin deleted from the genome of an RNA virus.

The intercistronic region between the maturation and coat-protein genes of RNA phage MS2 contains important regulatory and structural information. The sequence participates in two adjacent stem-loop structures, one of which, the coat-initiator hairpin, controls coat-gene translation and is thus under strong selection pressure. We have removed 19 out of the 23 nucleotides constituting the intercistronic region, thereby destroying the capacity of the phage to build the two hairpins. The deletion lowered coat-protein yield more than 1000-fold, and the titer of the infectious clone carrying the deletion dropped 10 orders of magnitude as compared with the wild type. Two types of revertants were recovered. One had, in two steps, recruited 18 new nucleotides that served to rebuild the two hairpins and the lost Shine-Dalgarno sequence. The other type had deleted an additional six nucleotides, which allowed the reconstruction of the Shine-Dalgarno sequence and the initiator hairpin, albeit by sacrificing the remnants of the other stem-loop. The results visualize the immense genetic repertoire created by, what appears as, random RNA recombination. It would seem that in this genetic ensemble every possible new RNA combination is represented.

Evolutionary relationships of the coelacanth, lungfishes, and tetrapods based on the 28S ribosomal RNA gene.

The origin of land vertebrates was one of the major transitions in the history of vertebrates. Yet, despite many studies that are based on either morphology or molecules, the phylogenetic relationships among tetrapods and the other two living groups of lobe-finned fishes, the coelacanth and the lungfishes, are still unresolved and debated. Knowledge of the relationships among these lineages, which originated back in the Devonian, has profound implications for the reconstruction of the evolutionary scenario of the conquest of land. We collected the largest molecular data set on this issue so far, about 3,500 base pairs from seven species of the large 28S nuclear ribosomal gene. All phylogenetic analyses (maximum parsimony, neighbor-joining, and maximum likelihood) point toward the hypothesis that lungfishes and coelacanths form a monophyletic group and are equally closely related to land vertebrates. This evolutionary hypothesis complicates the identification of morphological or physiological preadaptations that might have permitted the common ancestor of tetrapods to colonize land. This is because the reconstruction of its ancestral conditions would be hindered by the difficulty to separate uniquely derived characters from shared derived characters in the coelacanth/lungfish and tetrapod lineages. This molecular phylogeny aids in the reconstruction of morphological evolutionary steps by providing a framework; however, only paleontological evidence can determine the sequence of morphological acquisitions that allowed lobe-finned fishes to colonize land.

Potentiation of the bioavailability of daidzin by an extract of Radix puerariae.

The dose effect of pure daidzin on the suppression of ethanol intake in Syrian golden hamsters was compared with that of crude daidzin contained in a methanol extract of Radix puerariae (RP). EC50 values estimated from the graded dose-response curves for pure daidzin and RP extract daidzin are 23 and 2.3 mg per hamster per day, respectively. Apparently the antidipsotropic activity of the RP extract cannot be accounted for solely by its daidzin content (22 mg/g). In addition to daidzin, six other isoflavones were identified in the RP extract and quantified--namely, puerarin (160 mg per g of extract), genistin (3.7 mg/g), daidzein (2.6 mg/g), daidzein-4',7-diglucoside (1.2 mg/g), genistein (0.2 mg/g), and formononetin (0.16 mg/g). None of these, administered either alone or combined, contributes in any significant way to the antidipsotropic activity of the extract. Plasma daidzin concentration-time curves determined in hamsters administered various doses of pure daidzin or RP extract by i.p.injection indicate that the crude extract daidzin has approximately 10 times greater bioavailability than the pure compound. Reconstruction of the dose-response effects for pure and crude daidzin using bioavailable daidzin rather than administered dose gives a single curve. Synthetic daidzin added to the RP extract acquires the bioavailability of the endogenous daidzin that exists naturally in the extract. These results show that (i) daidzin is the major active principle in methanol extracts of RP, and (ii) additional constituents in the methanol extract of RP assist uptake of daidzin in golden hamsters.

Three-dimensional image reconstruction of dephosphorylated smooth muscle heavy meromyosin reveals asymmetry in the interaction between myosin heads and placement of subfragment 2

Regulation of the actin-activated ATPase of smooth muscle myosin II is known to involve an interaction between the two heads that is controlled by phosphorylation of the regulatory light chain. However, the three-dimensional structure of this inactivated form has been unknown. We have used a lipid monolayer to obtain two-dimensional crystalline arrays of the unphosphorylated inactive form of smooth muscle heavy meromyosin suitable for structural studies by electron cryomicroscopy of unstained, frozen-hydrated specimens. The three-dimensional structure reveals an asymmetric interaction between the two myosin heads. The ATPase activity of one head is sterically “blocked” because part of its actin-binding interface is positioned onto the converter domain of the second head. ATPase activity of the second head, which can bind actin, appears to be inhibited through stabilization of converter domain movements needed to release phosphate and achieve strong actin binding. When the subfragment 2 domain of heavy meromyosin is oriented as it would be in an actomyosin filament lattice, the position of the heads is very different from that needed to bind actin, suggesting an additional contribution to ATPase inhibition in situ.