Generic realignment in primuloid families of the Ericales s.l.: a phylogenetic analysis based on DNA sequences from three chloroplast genes and morphology

The phylogenetic interrelationships in Primulaceae, Myrsinaceae, and Theophrastaceae were investigated using DNA sequence data from the chloroplast genes atpB, ndhF, and rbcL. The three genes were analyzed separately, together, and in combination with morphology, using parsimony jackknifing. The sequence data are further explored by analyses of first and second codon position only, third positions only, and transversions only. The results show that all codon positions contribute group support to the ndhF tree, whereas third codon positions provide most of the structure in the atpB and rbcL trees. Analyzed separately, transversions in atpB and rbcL have little structure, whereas in ndhF they produce a well-resolved tree. We conclude that the most informative and robust results are obtained from analyses with all codon positions included and that the tree resulting from the combined analysis of all available data provides the best estimate of phylogeny.The results show that Maesa is sister to all other taxa from the three families. Theophrastaceae are well supported, but both Myrsinaceae and Primulaceae are paraphyletic. We conclude that four families should be recognized, Maesaceae, Theophrastaceae, Primulaceae, and Myrsinaceae. For all families to be monophyletic, Samolus was transferred to Theophrastaceae, and Lysimachia, Anagallis, Trientalis, Glaux, Asterolinon, and Pelletiera were moved to the Myrsinaceae together with the genera Coris, Ardisiandra, and Cyclamen.     

Seasonal and geographical differences in cleaner fish activity in the Mediterranean Sea

Investigations into symbioses may be important in order to analyse the grade of stability in ecosystems. Host–cleaner relationships were investigated in two localities of the Mediterranean Sea: Giglio (Tuscany, Italy) and Banyuls-sur-Mer (France). The cleaner wrasse, Symphodus melanocercus, was the main cleaner. Supplementary cleaners, such as the young of several wrasses, Symphodus mediterraneus, Symphodus ocellatus, Symphodus tinca, Coris julis and Ctenolabrus rupestris, are able to help out in times when there is a shortage of cleaners. Differences between the localities were obvious by a greater fish (host) density in Banyuls, which is probably due to eutrophication and might improve and increase cleaner activities. Regarding two seasons, spring and late summer, the fishes presented a lower degree of activity in Giglio, but a higher one in Banyuls – in late summer compared with spring. The main hosts were Chromis chromis, Symphodus tinca and Coris julis; additionally Diplodus sargus in Banyuls and Apogon imberbis in Giglio. Chromis chromis may be a key host species because of its distribution in large groups in the whole Mediterranean; Coris julis is of similar abundance. Symphodus melanocercus additionally came into contact with Symphodus tinca in order to partake of the food of the peacock wrasse. During cleaning activities, Diplodus sargus and other sparids present hesitant behaviour, where the cleaner probably picks more than parasites (skin, scales?) from the body surface. Apogon imberbis (which prefers dark habitats such as caves) totally rejects the main cleaner but was cleaned exclusively by Coris julis which, unlike Symphodus melanocercus, is regularly present near cave entrances. The investigations may present models of the evolutionary processes on specialised partnerships in ecosystems. Received in revised form: 10 August 2001 Electronic Publication     

Genetic study of Coris julis (Osteichthyes, Perciformes, Labridae) evolutionary history and dispersal abilities

Microsatellite markers have been used to study the genetic variability of rainbow wrasse (Coris julis) Mediterranean and Atlantic populations. Differentiation tests failed to reveal any significant genetic differentiation among samples from continental Portugal and the Azores, despite more than 1800 km of geographical separation. Preliminary results tended to indicate a significant genetic differentiation among Atlantic and Mediterranean samples. It also supported the specific status of Cape Verde populations (Coris atlantica). We compare these results with previous mtDNA analyses and propose a biogeographic scenario that could explain our results.     

Activation and repression sequences determine the lens-specific expression of the rat gamma D-crystallin gene.

Rat lens nuclear extracts contain a factor that binds to position -57 to -46 of the rat gamma D-crystallin promoter region. This factor protects the sequence 5'-CTGCCAACGCAG-3' in a footprint analysis. Binding to this region is crucial for maximal promoter activity in rat lens cells, but this sequence was unable to act as an enhancer when cloned in front of a heterologous promoter. A region directly upstream from this activating sequence, between position -85 to -67, acts as a strong silencer of promoter activity in non-lens cells. This silencing effect is mediated by trans-acting factor(s). Our data provide evidence for two regulatory elements in rat gamma D-crystallin gene expression, an activating sequence active in lens cells and a silencing sequence active only in non-lens cells. The factor that binds to the activating sequence could be detected only in lens cells and may be a determinant of the lens-specific expression of the gamma-crystallin genes.     

Different somatostatin receptor subtypes are operating in the brain of the teleost fish, Coris julis

Characterization of somatostatinergic (sst) neuronal activity through the application of nonpeptidyl agonists L-779,976 and L-817,818 which are highly specific for the sst receptors (sstr) sstr(2) and sstr(5), respectively, shows for the first time that sstr2, 5-like subtypes are the two major sstr subtypes operating in the brain of the teleost sea wrasse, Coris julis. A somewhat high but heterogeneous distribution pattern (> 30 < 180 fmol/mg wet tissue weight) of neurons expressing sstr2, 5 was reported in the different diencephalic regions plus in mesencephalon and telencephalon while low values were obtained in the cerebellum. Application of the above nonpeptidyl agonists permitted us to identify sstr2-like as the predominant subtype in telencephalic areas such as the entopeduncular nucleus (E) and postcommissural nucleus of the ventral telencephalon (Vp) as well as in hypothalamic and thalamic areas. At the same time high levels of neurons expressing sstr5-like, that greatly overlap those of sstr2-like in the diencephalic areas such as the anteroventral part of the preoptic nucleus (NPOav), the dorsal habenular nucleus (NHd) and the ventrolateral thalamic nucleus (VL), indicate that sstr2-like is very likely not the only sstr subtype acting in this fish brain. The predominance of sstr5-like in other brain areas is confirmed by the high quantities of this subtype in mesencephalic areas such as the torus longitudinalis (TLo). Overall, the discriminately differing densities of neurons expressing both subtypes seem to point to this system as a key molecular basis accounting for the distinct neurophysiological and behavioral sst-dependent activities in Coris julis.     

The supramedullary cells of the teleost Coris julis (L.): a noradrenergic neuronal system

This study, carried out on Coris julis (Labridae), is a contribution to the immunocytochemical characterization of fish supramedullary neurons. The significance of these giant cells has been debated since the beginning of the twentieth century. Our research provides the first evidence for a noradrenergic feature of this neuronal system. The possible role of supramedullary neurons as components of the autonomic nervous system is discussed. Moreover, the present results, taken together with our previous studies, surmise that this the first known case of colocalization of a neuropeptide (gastrin/CCK-like) and noradrenaline in the nervous system of teleosts.     

Sex-inversion of the hermaphroditic, protogynous teleost Coris julis L. (Labridae)

Sex-inversion of Coris julis , a protogynous hermaphrodite, was studied using histological and cytological criteria. Four stages were recognized: oocyte and oogonial atresia; occurrence of spermatogonia with primordial germ cells (PGC) in the ovarian wall; onset of spermatogonial proliferation; considerable proliferation of spermatogonia and PGC with build-up of seminiferous tubules. Spermatogonia arise from PGC, undifferentiated and bipotential cells, in which mitotic activity was detected.     

Histological and ultrastructural studies on the effects of hCG on sex inversion in the protogynous teleost Coris julis

In the protogynous diandric teleost Coris julis , sex inversion can be induced by injection of human chorionic gonadotropin (hCG). The histological changes of the gonad are accompanied by a transformation of the female livery to that of secondary males. At the ultrastructural level, the presence of intragonadal primordial germ cells in spermatogonial nests and within the newly forming seminiferous lobules is shown. Leydig cells and granulocytes appear in the inverting gonads. The results support the assumption that gonadotropic hormone has a key role in protogynous sex change.     

Searching for and predicting the activity of sites for DNA binding proteins: compilation and analysis of the binding sites for Escherichia coli integration host factor (IHF).

An analysis of the sequence information contained in a compilation of published binding sites for E. coli integration host factor (IHF) was performed. The sequences of twenty-seven IHF sites were aligned; the base occurrences at each position, the information content, and an extended consensus sequence were obtained for the IHF site. The base occurrences at each position of the IHF site were used with a program written for the Apple Macintosh computers in order to determine the similarity scores for published IHF sites. A linear correlation was found to exist between the logarithm of IHF binding and functional data (relative free energies) and similarity scores for two groups of IHF sites. The MacTargsearch program and its potential usefulness in searching for other sites and predicting their relative activities is discussed.     

cDNA sequence of the beta 2-subunit of human liver alcohol dehydrogenase

A cDNA library of mRNA from a human liver expressing the beta 2-subunit of alcohol dehydrogenase was constructed in lambda gt11. One clone coding for 352 of a total of 374 amino acid residues of the beta 2-subunit was isolated. The sequence differed from that of the beta 1-subunit at one nucleotide position resulting in an Arg/His exchange at position 47 of the peptide chain, in agreement with data from protein sequence analysis [(1984) FEBS Lett. 173, 360-366].     

Isolation of a fourth cysteinyl-containing peptide of the alpha-subunit of the F1 ATPase from Escherichia coli necessitates revision of the DNA sequence

The rapid determination of cysteinyl residues by Creighton's method [(1980) Nature 284, 487-489] led to the discovery of a discrepancy between protein and DNA sequence data in the alpha-subunit of the F1 ATPase from Escherichia coli [(1984) Arch. Biochem. Biophys. 229, 320-328]. We have isolated a cysteinyl-containing decapeptide from the alpha-subunit with a protein sequence (AGCAMGEYFR) which is only partially recognizable from DNA data. Re-sequencing of DNA in the region coding for the peptide has resulted in two corrections: insertion of a cytosine before position 715 and deletion of a thymine at position 731 of the uncA gene.     

Social organization,patterns of sexuality,and behavior of the wrasse Coris dorsomaculata at Miyake-jima,Japan

Synopsis The wrasse Coris dorsomaculata was investigated at Miyake jima, Japan. This species is sexually dichromatic and males arise from sex-changed females. Individuals were found living in harems which may be maintained through the defense of food by dominant males. The behavior of this species was similar to other studied wrasses, with the exception of a post-spawning display by males and the regular formation of multi-male aggregations in the late afternoon. Reproductive activity appeared to be affected by the warm-temperate climate at the study area. When day length was long, the spawning time coincided with outgoing tidal currents. When day length was short, the daily spawning time became fixed.     

Determination of the site of phosphorylation of nodulin 26 by the calcium-dependent protein kinase from soybean nodules.

Nodulin 26 is a nodule-specific protein that is associated with the symbiosome membrane of soybean root nodules. Nodulin 26 is an endogenous substrate for a novel calcium-dependent protein kinase (CDPK) of soybean root nodules. By phosphopeptide mapping of endoproteinase Lys-C-digested nodulin 26 and automated and manual peptide sequence analyses, we have identified the site on nodulin 26 phosphorylated by CDPK. We have also established that the phosphorylation site of nodulin 26 is identical to the phosphorylation site of CK-15, a synthetic peptide with the carboxyl-terminal sequence of nodulin 26. The phosphorylation of nodulin 26 occurs at position Ser262, and the phosphorylation of CK-15 occurs at the analogous position, Ser,6 in vitro. Thus, the CK-15 sequence apparently contains sufficient structural features of the phosphorylation site of nodulin 26 to be recognized by CDPK. On the basis of peptide mapping analysis of nodulin 26 from nodules that are metabolically labeled with [32P]phosphate, it appears that the site of nodulin 26 that is phosphorylated in vitro is also labeled in vivo. The data indicate that the carboxyl terminus of nodulin 26 is phosphorylated by CDPK and provide initial sequence data for the phosphorylation site of an endogenous substrate for a plant CDPK.     

Acid phosphatase activity in the supramedullary neurons of Coris julis (L.).

This work describes the acid phosphatase activity in supramedullary neurons of Coris julis, analyzed by a cytochemical method. The presence of both acid phosphatase-positive and -negative membrane bound granules indicates that only a part of the numerous electrodense granules in the supramedullary neurons can be interpreted as lysosomes. The great number of lysosomes in these large neurons of young animals is indicative of the rapid turnover of cell structures, which may be correlated with the high rate of synthesis. The electrodense granules showing no acid phosphatase activity are postulated to be vesicles containing gastrin/CCK-like peptide or precursors of this neuromediator.     

Structural basis for the species selectivity of a fibrin-specific monoclonal antibody

The structural determinant underlying the species specificity of a monoclonal anti-fibrin antibody (59D8) is the leucyl residue at position 5 in beta-chains of human fibrin. Anti-fibrin antibody 59D8 which had been elicited by immunization with human beta(1-7) peptide, Gly-His-Arg-Pro-Leu-Asp-Lys, binds to human and canine fibrins but not to bovine, ovine, or porcine fibrins. A comparison of the available amino acid sequence data suggested that the ability of anti-fibrin antibody 59D8 to discriminate among various fibrin beta-chains might be due to the amino acid at position 5. This was confirmed by competitive inhibition studies using synthetic fibrin-like peptides and determination of the amino acid sequences of the N-termini of ovine and porcine fibrin beta-chains. Edman degradation employing o-phthalaldehyde blocking permitted use of fibrin monomer rather than its separated constituent polypeptide chains. The same sequencing strategy was used to obtain partial sequence data for the alpha-chains of bovine, ovine, and porcine fibrin.     

Diagnostic accuracy of direct agglutination test,rK39 ELISA and six rapid diagnostic tests among visceral leishmaniasis patients with and without HIV coinfection in Ethiopia

Diagnosis of a first-time visceral leishmaniasis (VL) infection in Ethiopia is established by use of a rapid diagnostic test (RDT) detecting antibodies against rK39, direct agglutination test (DAT) and microscopy according to the national algorithm. The performance of individual tests and algorithm is variable and depends on several factors, one being HIV status. Limited data are available on the performance of tests in VL-HIV coinfected patients.Assessment of the performance of DAT (ITM-A), rK39 ELISA (Serion) and six RDT (Onsite Leishmania Ab CTK, Antigen ICT Xinjier, IT Leish Biorad, Kalazar Detect Inbios, rK39 IgG1 Coris, rk28 IgG1 Coris) for the diagnosis of VL was done on a panel of 91 stored serum and plasma samples of ‘first-episode’ suspected VL patients, with HIV coinfection (n = 51) and without (n = 40). A combined reference standard was used: either positive microscopy on tissue aspirates, or in case of negative microscopy, positive PCR results on the aspirate slide. Additionally, endemic healthy controls (n = 20), non-endemic controls (n = 10) and patients with confirmed malaria infection (n = 10) were tested for specificity evaluation. Sensitivities ranged from 69.2% for DAT (applied cut-off ≥ 1/3200) to 92.2% for the Onsite RDT, whereas specificities ranged from 20.0% for Kalazar Antigen ICT to 100% for IT Leish and rK39 IgG1. Sensitivities from all assays decreased upon stratification according to HIV status but was only significantly different for rK39 Serion ELISA (p-value 0.0084) and the Onsite RDT (p-value 0.0159).In conclusion, performance of commercially available assays for VL on samples from Northern-Ethiopian patients varied widely with a substantial decrease in sensitivity in the VL-HIV coinfected group. Clear guidelines on minimal performance criteria of individual tests and algorithms are needed, as well as which reference standard should be used to determine the performance.     

Amino acid sequences of hemoglobins I and II from root nodules of the non-leguminous Parasponia rigida-rhizobium symbiosis, and a correction of the sequence of hemoglobin I from Parasponia andersonii

The amino acid sequence of hemoglobins I (pI 6.15 as oxyhemoglobin) and II (pI 5.64 as oxyhemoglobin) from the nitrogen-fixing root nodules of Parasponia rigida have been determined by protein sequencing. The sequence of hemoglobin I (pI 6.16, as oxyhemoglobin) from Parasponia andersonii was re-examined and the corrected primary structure, now in agreement with that predicted from the DNA sequence, is reported. The three Parasponia hemoglobins contain 161 amino acid residues (Mr approximately equal to 18,700 including the heme) with a single cysteine residue and five methionine residues. The N-terminal serine is blocked by an acetyl group. The primary structure of the Parasponia hemoglobins is highly conserved. Hemoglobins I from the two species of Parasponia are identical; both show microheterogeneity at position 30 (Asp/Glu substitution) and hemoglobin I from P. rigida shows microheterogeneity at position 150 (Ala/Val) while hemoglobin I from P. andersonii has only an Ala at 150. P. rigida hemoglobin II shows no microheterogeneity at these positions, having Asp and Val residues respectively, and it contains a single amino acid change of a Gln for an Arg at position 85, which accounts for the 0.5 unit difference in isoelectric point observed between hemoglobins I and II. The sequence data are consistent with allelic heterogeneity at a single locus rather than different genes.     

Estimating the reliability of evolutionary trees

Six protein sequences from the same 11 mammalian taxa were used to estimate the accuracy and reliability of phylogenetic trees using real, rather than simulated, data. A tree comparison metric was used to measure the increase in similarity of minimal trees as larger, randomly selected subsets of nucleotide positions were taken. The ratio of the observed to the expected number of incompatibilities for each nucleotide position (character) is a good predictor of the number of changes required at that position on the minimal (most-parsimonious) tree. This allows a higher weighting of nucleotide positions that have changed more slowly and should result in the minimal length tree converging to the correct tree as more sequences are obtained. An estimate was made of the smallest subset of trees that need to be considered to include the actual historical tree for a given set of data. It was concluded that it is possible to give a reasonable estimate of the reliability of the final tree, at least when several sequences are combined. With the present data, resolving the rodent- primate-lagomorph (rabbit) trichotomy is the least certain aspect of the final tree, followed then by establishing the position of dog. In our opinion, it is unreasonable to publish an evolutionary tree derived from sequence data without giving an idea of the reliability of the tree.      

Primary structure of scorpion anti-insect toxins isolated from the venom of Leiurus quinquestriatus quinquestriatus

The amino acid sequences of insect-selective scorpion toxins, purified from the venom of Leiurus quinquestriatus quinquestriatus, have been determined by automatic phenyl isothiocyanate degradation of the S-carboxymethylated proteins and derived proteolytic peptides. The excitatory toxin Lqq IT1 and Lqq IT1' (70 residues) show the shift of one half-cystine from an external position, which is characteristic of anti-mammal toxins, to an internal sequence position. Lqq IT2 (61 residues) displays the half-cystine residue in position 12, common to the sequence of all known anti-mammal toxins; it induces flaccid paralysis on insects but is non-toxic for the mouse. Lqq IT2 structurally defines a new type of anti-insect toxins from scorpion venoms. CD spectra and immunological data are in agreement with this finding.     

The juxtamembrane sequence of cytochrome P-450 2C1 contains an endoplasmic reticulum retention signal

The N-terminal signal anchor of cytochrome P-450 2C1 mediates retention in the endoplasmic reticulum (ER) membrane of several reporter proteins. The same sequence fused to the C terminus of the extracellular domain of the epidermal growth factor receptor permits transport of the chimeric protein to the plasma membrane. In the N-terminal position, the ER retention function of this signal depends on the polarity of the hydrophobic domain and the sequence KQS in the short hydrophilic linker immediately following the transmembrane domain. To determine what properties are required for the ER retention function of the signal anchor in a position other than the N terminus, the effect of mutations in the linker and hydrophobic domains on subcellular localization in COS1 cells of chimeric proteins with the P-450 signal anchor in an internal or C-terminal position was analyzed. For the C-terminal position, the signal anchor was fused to the end of the luminal domain of epidermal growth factor receptor, and green fluorescent protein was additionally fused at the C terminus of the signal anchor for the internal position. In these chimeras, the ER retention function of the signal anchor was rescued by deletion of three leucines at the C-terminal side of its hydrophobic domain; however, deletion of three valines from the N-terminal side did not affect transport to the cell surface. ER retention of the C-terminal deletion mutants was eliminated by substitution of alanines for glutamine and serine in the linker sequence. These data are consistent with a model in which the position of the linker sequence at the membrane surface, which is critical for ER retention, is dependent on the transmembrane domain.