Testing the accuracy of methods for reconstructing ancestral states of continuous characters.

Many methods are available for estimating ancestral values of continuous characteristics, but little is known about how well these methods perform. Here we compare six methods: linear parsimony, squared-change parsimony, one-parameter maximum likelihood (Brownian motion), two-parameter maximum likelihood (Ornstein-Uhlenbeck process), and independent comparisons with and without branch-length information. We apply these methods to data from 20 morphospecies of Pleistocene planktic Foraminifera in order to estimate ancestral size and shape variables, and compare these estimates with measurements on fossils close to the phylogenetic position of 13 ancestors. No method produced accurate estimates for any variable: estimates were consistently less good as predictors of the observed values than were the averages of the observed values. The two-parameter maximum-likelihood model consistently produces the most accurate size estimates overall. Estimation of ancestral sizes is confounded by an evolutionary trend towards increasing size. Shape showed no trend but was still estimated very poorly: we consider possible reasons. We discuss the implications of our results for the use of estimates of ancestral characteristics.     

Purification and characterization of a lysophospholipase from human amnionic membranes

We have identified the presence of a lysophospholipase in human placental tissues and have purified this enzyme from the amnion. The specific activity was highest in the amnion and decreased across adjacent tissues. The purification involved the use of DEAE-Sephadex, phenyl-Sepharose, hydroxylapatite, and sulfylpropyl Sephadex chromatography. The activity of the purified enzyme toward palmitoyl lysophosphatidylcholine is 2.5 mumol min-1 mg-1 and the pH optimum is 7.0. The enzyme is not inhibited by EDTA and does not appear to have a metal ion requirement. The enzyme may be of membrane origin; the purified enzyme requires the presence of detergent during storage. The effects of substrate composition and physical state on enzymatic activity were explored. The enzyme was not active toward mono-, di-, or triglycerides, nor toward diacyl phospholipid. The enzyme was active toward myristoyl and palmitoyl lysophosphatidylcholine at concentrations where these substrates spontaneously form micelles or where Triton X-100 was used to induce co-micellization of the substrate at low concentrations with detergent. A role for this enzyme in processing the lysophospholipid product of phospholipase A action must be considered in evaluating arachidonic acid production in human fetal membranes and placental tissue, particularly during the initiation of labor.     

Impaired calcium mobilisation in the 7315a prolactin-secreting pituitary tumour

The 7315a tumour secretes prolactin, but is refractory to enhancement of prolactin release by thyrotrophin-releasing hormone (TRH). In order to investigate further this refractoriness of the 7315a tumour cell, we compared cells from the tumour and from the normal pituitary with regard to TRH-enhanced fractional 45Ca2+ efflux and inositol phosphate production. TRH caused a large efflux of calcium from normal pituitary cells, but only mildly enhanced calcium efflux from the tumour cells. In contrast, TRH enhanced total inositol phosphate generation in both groups of cells to a similar degree. We therefore conclude that prolactin release from 7315a tumour cells is refractory to TRH due, at least in part, to impaired mobilisation of intracellular calcium by inositol phosphates.     

Bacteriophage Resistance Plasmid pTR2030 Inhibits Lytic Infection of r(1)t Temperate Bacteriophage but Not Induction of r(1)t Prophage in Streptococcus cremoris R1

The effects of pTR2030 on the replication of four small isometric bacteriophages were examined in Streptococcus cremoris R1. Three lytic phages (652, 720, and 751), which were isolated independently over a 29-year period, were unable to form plaques on a pTR2030 transconjugant of S. cremoris R1. The fourth phage evaluated, phage r(1)t, was a temperate phage induced from S. cremoris R1 by treatment with mitomycin C. A prophage-cured derivative of S. cremoris R1, designated R1Cs, was isolated and served as a lytic indicator for phage r(1)t. Strain R1Cs and a derivative of this strain that was relysogenized with r(1)t, designated R1Cs(r(1)t), were used as conjugal recipients for transfer of the phage resistance plasmid pTR2030. pTR2030 transconjugants of strains R1Cs and R1Cs(r(1)t) were evaluated for sensitivity to r(1)t phage and induction of r(1)t prophage, respectively. The temperate phage r(1)t adsorbed eficiently but did not form plaques on the prophage-cured, pTR2030 transconjugant strain T-R1Cs. However, in the r(1)t lysogen [T-R1Cs(r(1)t)], pTR2030 did not inhibit prophage induction with mitomycin C, cell lysis, or production of infective r(1)t phage particles. The data demonstrated that pTR2030-induced resistance inhibited lytic infection by r(1)t phage from without but did not retard lytic development after prophage induction within the cell. It was suggested that pTR2030-encoded phage resistance to small isometric phages may, therefore, act at the cell surface or membrane to prevent phage DNA passage into the host cell or inhibit early events required for lytic replication of externally infecting phage.     

A novel wheat alpha-amylase gene (alpha-Amy3)

Summary A genomic clone of a wheat -amylase gene (Amy3/33) was identified, on the basis of hybridisation properties, as different from -Amy1 and -Amy2 genes which had been characterised previously. The nucleotide sequence revealed that this gene has the normal sequence motifs of an active gene and an open reading frame interrupted by two introns. The protein sequence encoded by this open reading frame is recognisably similar to that of -amylase from the -Amy1 and -Amy2 genes and there is high sequence homology in all three proteins at the putative active sites and Ca⁺⁺ binding region. In addition, the introns are at positions equivalent to the position of introns in the -Amy1 and -Amy2 genes. However, the sequence was less similar to -Amy1 and -Amy2 than these are to each other. Southern blot analysis showed that the Amy3/33 DNA is one of a small multigene family carried on a different chromosome (group 5) from either the -Amy1 or -Amy2 genes. A further difference from the -Amy1 and -Amy2 genes was the pattern of expression. Amy3/33 was expressed only in immature grains and, unlike the -Amy1 and -Amy2 genes, not at all in germinating aleurones. These data suggested therefore that this gene represents a third type of -amylase gene, not described before, which shares a common evolutionary ancestor with the -Amy1 and -Amy2 genes.     

Differences in the chemical and catalytic characteristics of two crystallographically ''identical'' enzyme catalytic sites. Characterization of actinidin and papain by a combination of pH-dependent substrate catalysis kinetics and reactivity probe studies targeted on the catalytic-site thiol group and its immediate microenvironment.

The characteristics of actinidin (EC 3.4.22.14) and papain (EC 3.4.22.2), two cysteine proteinases whose catalytic-site regions appear to superimpose to a degree that approaches atomic co-ordinate accuracy of both crystal structures, were evaluated by determining (a) the pH-dependence in acid media of the acylation process of the catalytic act (k+2/Ks) using N alpha-benzoyl-L-arginine p-nitroanilide (L-Bz-Arg-Nan) as substrate and (b) the sensitivity of the reactivity of the catalytic-site thiol group and its pH-dependence to structural change in small, thiol-specific, two-protonic-state reactivity probes (2,2'-dipyridyl disulphide and methyl 2-pyridyl disulphide) where enzyme-probe contacts should be restricted to areas close to the catalytic site. Distortion of the catalytic sites of the two enzymes at pH less than 4 was evaluated over time-scales appropriate for both stopped-flow reactivity probe kinetics (less than or equal to 1-2 s) and steady-state substrate catalysis kinetics (3-5 min) by using the 2,2'-dipyridyl disulphide monocation as a titrant for non-distorted catalytic sites. This permitted a lower pH limit to be defined for valid kinetic analysis of both types. The behaviour of the enzymes at pH less than 4 requires a kinetic model in which the apparently biomolecular reaction of enzyme with probe reagent is separated from the process leading to loss of conformational integrity by a potentially reversible step. The acylation of actinidin with L-Bz-Arg-Nan in acidic media occurs in two protonic states, one produced by raising the pH across pKa less than 4 which probably characterizes the formation of -S-/-ImH+ ion pair (pKa approx. 3) and the other, of higher reactivity, produced by raising the pH across pKa 5.5, which may characterize rearrangement of catalytic-site geometry. The pH-dependence of the acylation of papain by L-Bz-Arg-Nan is quite different and is not influenced by protonic dissociation with pKa values in the range 5-6. The earlier conclusion that the acylation of papain depends on two protonic dissociations each with pKa approx. 4 was confirmed. This argument is now more firmly based because titration with 2,2'-dipyridyl disulphide permits the loss of conformational integrity to be taken into account in the analysis of the kinetic data at very low pH. Methyl 2-pyridyl disulphide was synthesized by reaction of pyridine-2-thione with methyl methanethiolsulphonate and its pKa at I = 0.1 was determined by spectral analysis at 307 nm to be 2.8.(ABSTRACT TRUNCATED AT 400 WORDS)