Inferring the number of evolutionary events from DNA coding sequence differences

The estimation of the amount of evolutionary divergence that has taken place between two DNA coding sequences depends strongly on the degree of constraint on amino acid replacements. If amino acid replacements are relatively unconstrained, the individual nucleotide is the appropriate unit of analysis and the method of Tajima and Nei can be used. If amino acid replacements are constrained, however, this method is shown to be inapplicable. For sequences with strong amino acid constraints, a method is outlined analogous to the Tajima and Nei method using codons as the unit of analysis. Only synonymous substitutions are used. Codon usage data can be employed to estimate the necessary parameters of the calculation, or a priori models of substitution may be employed. Sequences with significant but intermediate constraints on amino acid replacements are, in principle, unanalyzable.      

Structure and tissue-specific developmental expression of a sea urchin arylsulfatase gene

Arylsulfatases are a group of enzymes that remove sulfate moieties from a diverse set of substrates including glycoproteins, steroids, and cerebrosides. We have isolated recombinant cDNA clones corresponding to an arylsulfatase (SpARS) message that encodes an abundant protein of pluteus larvae of the sea urchin Strongylocentrotus purpuratus. Although vertebrate arylsulfatases have broad tissue distributions, in situ hybridization with a probe for SpARS shows that the sea urchin message accumulates in the embryo only in the single cell type of aboral ectoderm and its precursors. The message is first detectable by RNase protection assays around hatching blastula stage and accumulates through pluteus larva stage. The open reading frame of cDNA clones is 1701 nt long and encodes a deduced protein with a predicted molecular mass of 61 kDa. Analysis of corresponding genomic DNA clones reveals that the pre-mRNA contains six exons. Consistent with the fact that arylsulfatase enzyme activity is extracellular, this polypeptide has a hydrophobic leader sequence and three potential glycosylation sites. Furthermore, hybridization in situ shows that in blastulae arylsulfatase message is preferentially concentrated around nuclei at the basal sides of cells. The S. purpuratus sequence is very similar to that recently reported for the same enzyme from Hemicentrotus pulcherrimus and 30% of the amino acid residues are also identical to those of both human arylsulfatase C (steroid sulfatase) and arylsulfatase A. Sequence relationships among these four mRNAs suggest that, assuming equal rates of evolution, the duplication separating the human genes occurred at about the time of separation of the echinoderm and vertebrate lineages.     

Detection of poly A+ RNA in sea urchin eggs and embryos by quantitative in situ hybridization

We present an improved procedure for detecting poly A tracts in situ by hybridization of 3H poly U. Glutaraldehyde fixation achieves significantly higher retention of RNA and better morphologic preservation than does Carnoy's. A dramatic increase in signal to noise is obtained by prehybridization treatment of glutaraldehyde-fixed sections with proteinase K and acetic anhydride. Measurement of the increase in poly A concentration after fertilization by solution titration and by in situ hybridization are in excellent agreement indicating that in situ measurements yield accurate relative estimates of local RNA concentrations in sections. Examination of the grain density distribution in section of sea urchin eggs and cleaving embryos reveals no major cytoplasmic localization of poly A+ RNA, although nuclei show much less labelling and micromeres of 16-cell embryos have a small, but significant, reduction in poly A concentration.     

Biological and biosynthetic properties of poly-l-malate

Abstract β-Poly- l -malate (PMA) is a highly soluble polyanion specifically synthesized during the plasmodial stage of the life cycle in Physarum polycephalum . The polymer partitions in a growth-dependent fashion between cell nuclei, the cytoplasm and the culture medium, where it is slowly hydrolyzed. It strongly interacts with certain soluble nuclear proteins suggesting the function as a mobile matrix involved in stock-piling of such molecules and required in the synchronization of the nuclear cell division typical for plasmodia. It is synthesized continuously over the cell cycle during growth involving a PMA polymerase that seems genetically related to the enzyme catalyzing in certain bacteria the polymerization of poly-β-hydroxybutyrate.     

Iron transport systems of Serratia marcescens.

Serratia marcescens W225 expresses an unconventional iron(III) transport system. Uptake of Fe3+ occurs in the absence of an iron(III)-solubilizing siderophore, of an outer membrane receptor protein, and of the TonB and ExbBD proteins involved in outer membrane transport. The three SfuABC proteins found to catalyze iron(III) transport exhibit the typical features of periplasmic binding-protein-dependent systems for transport across the cytoplasmic membrane. In support of these conclusions, the periplasmic SfuA protein bound iron chloride and iron citrate but not ferrichrome, as shown by protection experiments against degradation by added V8 protease. The cloned sfuABC genes conferred upon an Escherichia coli aroB mutant unable to synthesize its own enterochelin siderophore the ability to grow under iron-limiting conditions (in the presence of 0.2 mM 2.2'-dipyridyl). Under extreme iron deficiency (0.4 mM 2.2'-dipyridyl), however, the entry rate of iron across the outer membrane was no longer sufficient for growth. Citrate had to be added in order for iron(III) to be translocated as an iron citrate complex in a FecA- and TonB-dependent manner through the outer membrane and via SfuABC across the cytoplasmic membrane. FecA- and TonB-dependent iron transport across the outer membrane could be clearly correlated with a very low concentration of iron in the medium. Expression of the sfuABC genes in E. coli was controlled by the Fur iron repressor gene. S. marcescens W225 was able to synthesize enterochelin and take up iron(III) enterochelin. It contained an iron(III) aerobactin transport system but lacked aerobactin synthesis. This strain was able to utilize the hydroxamate siderophores ferrichrome, coprogen, ferrioxamine B, rhodotorulic acid, and schizokinen as sole iron sources and grew on iron citrate as well. In contrast to E. coli K-12, S. marcescens could utilize heme. DNA fragments of the E. coli fhuA, iut, exbB, and fur genes hybridized with chromosomal S. marcescens DNA fragments, whereas no hybridization was obtained between S. marcescens chromosomal DNA and E. coli fecA, fhuE, and tonB gene fragments. The presence of multiple iron transport systems was also indicated by the increased synthesis of at least five outer membrane proteins (in the molecular weight range of 72,000 to 87,000) after growth in low-iron media. Serratia liquefaciens and Serratia ficaria produced aerobactin, showing that this siderophore also occurs in the genus Serratia.     

3-Alkyl-2-phenylbenzo[b]thiophenes: Nonsteroidal estrogen antagonists with mammary tumor inhibiting activity

A number of 2-(4-hydroxyphenyl)benzo[b]thiophenes with a hydroxy group in position 5 or 6 and a short alkyl group at C-3 were synthesized and studied for their estrogen receptor affinities. Relative binding affinities (RBA) for the calf uterine estrogen receptor ranged from 3 to 60 (17β-estradiol = 100). The highest RBA values were found with ethyl derivatives [3 (5-OH): 60; 7 (6-OH): 28]. In accord with their receptor affinity, all benzothiophenes exhibited endocrine activity in the immature mouse uterine weight test. At doses of 0.25–7.0 mg/kg body weight, they showed partial estrogen antagonism and usually weak estrogenic effects. All compounds entered tests with hormone-sensitive human MCF-7 breast cancer cells. At concentrations of 1μM and higher, most of the derivatives displayed significant inhibition of cell growth. These results prompted us to test them in vivo for cytostatic activity using hormone-dependent MXT mouse mammary tumors. The 5-hydroxy derivatives 3 and 4 strongly inhibited the growth of these tumors. After 4 weeks of treatment with 3 × 4.2 mg/kg of compound 3, the average tumor weight was reduced by 83% vs control (tamoxifen at equimolar dose: 74%). The 6-hydroxy derivative 7 required higher doses (25 mg/kg) to give rise to the same effect. At the end of therapy, no increase of uterine weight due to an estrogenic effect was observed. We assume therefore that the antineoplastic activity of these compounds in this tumor model is due mainly to their estrogen antagonism.