Molecules,fossils, and the origin of tetrapods

Summary Since the discovery of the coelacanth, Latimeria chalumnae, more than 50 years ago, paleontologists and comparative morphologists have debated whether coelacanths or lungfishes, two groups of lobe-finned fishes, are the closest living relatives of land vertebrates (Tetrapoda). Previously, Meyer and Wilson (1990) determined partial DNA sequences from two conservative mitochondrial genes and found support for a close relationship of lungfishes to tetrapods. We present additional DNA sequences from the 12S rRNA mitochondria gene for three species of the two lineages of lungfishes that were not represented in the first study: Protopterus annectens and Protopterus aethiopicus from Africa and Neoceratodus forsteri (kindly provided by B. Hedges and L. Maxson) from Australia. This extended data set tends to group the two lepidosirenid lungfish lineages (Lepidosiren and Protopterus) with Neoceratodus as their sister group. All lungfishes seem to be more closely related to tetrapods than the coelacanth is. This result appears to rule out the possibility that the coelacanth lineage gave rise to land vertebrates. The common ancestor of lungfishes and tetrapods might have possessed multiple morphological traits that are shared by lungfishes and tetrapods [Meyer and Wilson (1990) listed 14 such traits]. Those traits that seem to link Latimeria and tetrapods are arguably due to convergent evolution or reversals and not to common descent. In this way, the molecular tree facilitates an evolutionary interpretation of the morphological differences among the living forms. We recommended that the extinct groups of lobe-finned fishes be placed onto the molecular tree that has lungfishes and not the coelacanth more closely related to tetrapods. The placement of fossils would help to further interpret the sequence of morphological events and innovations associated with the origin of tetrapods but appears to be problematic because the quality of fossils is not always high enough, and differences among paleontologists in the interpretation of the fossils have stood in the way of a consensus opinion for the branching order among lobefinned fishes. Marshall and Schultze (1992) criticized the morphological analysis presented by Meyer and Wilson (1990) and suggest that 13 of the 14 morphological traits that support the sister group relationship of lungfishes and tetrapods are not shared derived characters. Here we present further alternative viewpoints to the ones of Marshall and Schultze (1992) from the paleontological literature. We argue that all available information (paleontological, neontological, and molecular data) and rigorous cladistic methodology should be used when relating fossils and extant taxa in a phylogenetic framework. Offprint requests to: Axel Meyer     

Effects of Dopaminergic Transmission Interruption on the D2 Receptor Isoforms in Various Cerebral Tissues

We examined the effects of an interruption of dopamine neurotransmission, by either dopamine receptor blockade or degeneration of dopamine neurons by 6-hydroxydopamine, on the levels of D2 receptor mRNAs. In addition, we evaluated by the polymerase chain reaction (PCR) the relative abundance of the two D2 receptor isoform mRNAs generated by alternative splicing. Daily injections of 4 mg/kg of haloperidol to rats elicited in striatum a rapid and progressive increase in D2 receptor mRNA levels, which reached 70% after a 15-day treatment. By contrast, there was no apparent change in D2 receptor mRNA levels in cerebral cortex and pons-medulla, in spite of an increased density of D2 receptor in the former tissue. Using the PCR with primers flanking the alternative exon, we observed that the relative proportion of the shorter receptor isoform (D2S) mRNA was slightly but significantly enhanced in cerebral cortex (17%) and pons-medulla (18%) after a 15-day haloperidol treatment. Unilateral degeneration of dopamine neurons induced by local injection of 6-hydroxydopamine resulted in a marked decrease in levels of total D2 receptor mRNAs in substantia nigra (-79%) and ventral tegmental (-63%) area, two cell body areas. In the substantia nigra, the longer isoform (D2L) mRNA was significantly more decreased in content than the D2S isoform mRNA, so that there was a large enhancement in the relative abundance of the latter (81%).(ABSTRACT TRUNCATED AT 250 WORDS)     

The xylanase introns from Cryptococcus albidus are accurately spliced in transgenic tobacco plants

The xylanase gene from Cryptococcus albidus contains seven introns. Genomic and cDNA clones under the control of the CaMV 35S promoter were transferred into tobacco plants using Agrobacterium-mediated cell transformation. The genes were transcribed and the mRNAs were amplified by the polymerase chain reaction using primers on each side of the intron region. About 90% of the amplification products from plants transformed with the genomic clone corresponded to the size of the pre-mRNA (1.2 kb) and 10% represented the spliced product (0.85 kb). The 0.85 kb fragment was cloned and sequenced and the result indicated that the introns from the xylanase gene were accurately spliced by the plant cells.     

Low hydration phase properties of phospholipid mixtures

An experimental investigation of the low hydration phase properties of phospholipid mixtures is described. ²H (D₂O) NMR, X-ray diffraction and differential scanning calorimetry have been used to elucidate the phase properties of mixtures of the mixed chain phospholipids palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylethanolamine (POPE). At 10% hydration pure POPE exhibited a H_II phase above 330 K, a fluid lamellar phase below 315 K, and a minimally hydrated crystalline phase below 300 K. For the 1:1 mixture, the samples exhibited only gel or fluid phases between 270 K and 360 K for hydrations in the range 15% to 30%. Below 15% hydration the mixture exhibited two fluid phases with different repeat spacings, as predicted previously.     

Measurement of the exchange rates of rapidly exchanging amide protons: Application to the study of calmodulin and its complex with a myosin light chain kinase fragment

Summary A technique is described for measuring the approximate exchange rates of the more labile amide protons in a protein. The technique relies on a comparison of the intensities in¹H–¹⁵N correlation spectra recorded with and without presaturation of the water resonance. To distinguish resonance attenuation caused by hydrogen exchange from attenuation caused by cross relation, the experiment is repeated at several different pH values and the difference in attenuation of any particular amide resonance upon presaturation is used for calculating its exchange rate. The technique is demonstrated for calmodulin and for calmodulin complexed with its binding domain of skeletal muscle myosin light chain kinase. Upon complexation, increased amide exchange rates are observed for residues Lys⁷⁵ through Thr⁷⁹ located in the central helix of calmodulin, and for the C-terminal residues Ser¹⁴⁷ and Lys¹⁴⁸. In contrast, a decrease in amide exchange rate is observed at the C-terminal end of the F helix, from residues Thr¹¹⁰ through Glu¹¹⁴.Istituto Guido Donegani, Novara, Italy     

Hückel framework study on the superconducting vibronic interaction in (AB) N chains

Superconducting vibronic interaction in the vibronic superconductivity motif has been studied in the Hückel framework for (AB) _N chain systems. Within the on-site and nearest-neighbor approximation a new vibronic constant, /L, has been introduced, of which the importance has been discussed. The effect of the vibronic operator, , has also been studied. It is also concluded that the size-dependence of the superconducting vibronic interaction also exists in the (AB) _N chain systems.On leave from the Changchun Institute of Applied Chemistry, Academia Sinica, Changchun, P. R. China, as an STA fellowship awardee hosted by the Institute of Physical and Chemical Research of Japan.     

Expression of aChlamydia anticarbohydrate single-chain antibody as a maltose binding fusion protein

A single-chain antibody fragment has been constructed for an antibody that binds to theChlamydia specific carbohydrate structure of the lipopolysaccharide. Single-chain protein was expressed and secreted into the periplasmic space ofE. coli as a fusion protein with the maltose binding protein. The fusion protein was purified in one step by virtue of its ability to bind to maltose. In a sandwich ELISA, the eluted protein boundChlamydia lipopolysaccharide, which demonstrates that the single-chain protein domain will function as part of a fusion protein. The expression of maltose binding fusion proteins into the periplasmic space could be used for production of other single-chain antibodies or protein fragments requiring appropriate folding and disulfide bond formation.