Constructional morphology: The analysis of constraints in evolution dedicated to A. Seilacher in honour of his 60. birthday

Evolutionary change is opportunistic, but its course is strongly constrained in several fundamental ways. These constraints (historical/phylogenetic, functional/adaptive, constructional/morphogenetic) and their dynamic relationships are discussed here and shown to constitute the conceptual framework of Constructional Morphology. Notwithstanding recent published opinions which claim that the discovery of constraints renders Neodarwinian selection theory obsolete, we regard the insights of Constructional Morphology as being entirely consistent with this theory. As is shown here in the case of the Hyracoidea, formal analysis of the constraints which have framed the evolution of various characters extends our understanding of the evolution of a taxon.     

Catalysis by heteropentalenes of electron transfer to oxygen from ferredoxin-NADP oxidoreductase reduced by NADPH

A range of heteropentalene and bipyridinium compounds have been tested as catalysts of electron transfer to oxygen from spinach ferredoxin-NADP⁺ oxidoreductase reduced by NADPH. For a particular class of compound, the rate of oxygen reduction increased with increasing midpoint potential of the compound under conditions in which reduction of the compound was rate-limiting. Compounds with similar midpoint potentials from different structural classes showed marked differences in rate, attributed to specificity in the interaction with ferredoxin-NADP⁺ oxidoreductase.     

A Salmonella typhimurium strain defective in uracil catabolism and beta-alanine synthesis

A selection procedure for uracil catabolism mutant strains involving indicator dye plates was developed. Using this method, a strain defective in uracil catabolism has been isolated in Salmonella typhimurium that was temperature-sensitive at 42 degrees C where it required low concentrations of N-carbamoyl-beta-alanine, beta-alanine or pantothenic acid for growth. An extract of the mutant strain degraded uracil at 37 degrees C at a significantly diminished rate compared to that observed for the wild-type strain under the same growth conditions. The conversion of dihydrouracil to N-carbamoyl-beta-alanine was blocked at all temperatures examined in the mutant strain. By means of genetic analysis, the mutant strain was determined to be defective at two genetic loci. Transduction studies with bacteriophage P22 indicated that the panD gene is mutated in this strain, accounting for its beta-alanine requirement. Episomal transfers between Escherichia coli and the mutant strain provided evidence that the defect in uracil catabolism was located in another region of the S. typhimurium chromosome.     

Discrimination between the effects of X-ray irradiation of the mouse oocyte and uterus on the induction of dominant lethals and congenital anomalies. II. Localised irradiation experiments

In order to evaluate whether irradiation of the postimplantation maternal environment contributed to the induction of postimplantation mortality or congenital anomalies, mouse ovaries were surgically exteriorised and selectively irradiated or shielded in a specially constructed apparatus. The results show that exposure of the mouse abdomen and uterus to 3.70 Gy X-rays, 15-21 days prior to conception, has no significant effect on the incidence of either postimplantation mortality or congenital anomalies. Exposure of the ovaries to 3.27 Gy X-rays during the same period, however, increased the frequency of both postimplantation mortality and congenital anomalies.     

Reversal of the effects of aging in soybean seeds

Accelerated aging predisposed seeds to imbibition injury. Slowing the rate of hydration prevented the loss of germinability due to imbibition injury. Germinability of accelerated aged seeds (50 hours) was increased from 10 to 90% by controlling the rate of imbibition. Slow hydration also prevented seed electrolyte leakage. This may indicate that cell membrane permeability or rupture was a major factor contributing to the loss of germinability after aging.

Reversal of the effects of aging (repair) was accomplished by slowly inbibing and then redrying seeds (priming). This treatment lowered steep water conductivity by a factor of 2 to 5. Priming also increased the per cent germination of low vigor seeds. The mechanism of this reversal was probably metabolic because it depended on temperature, seed moisture, and treatment duration.

Priming doubled the survival of seeds in the accelerated aging vigor test. The `rejuvenation' was accepted as evidence for metabolic repair. Since the `vigor' of seeds was increased by priming, metabolic repair probably included other subcellular components as well as the plasma membrane.

     

Antagonistic effects of thyrotropin and epidermal growth factor on thyroglobulin mRNA level in cultured thyroid cells

Both thyrotropin (TSH) and epidermal growth factor (EGF) are potent mitogenic agents when added to dog thyroid cells in primary culture [Roger, P. P. and Dumont, J. E. (1984) Mol. Cell. Endocrinol. 36, 79-93]. The concomitant effect of these agents on the differentiation state of the cells was appreciated using cell morphology, iodide trapping, thyroglobulin synthesis and cytoplasmic thyroglobulin mRNA content as markers. Together with previous results [Mol. Cell. Endocrinol. 36, 79-93 (1984)] it is shown that cells cultured in the continuous presence of TSH maintain all the parameters at a near normal level. In the absence of TSH, thyroglobulin mRNA decreased to very low, though still detectable levels. Addition of TSH restored subnormal mRNA levels. Culture of cells in the presence of EGF for 4-6 days affected profoundly their morphology, abolished iodide trapping and decreased thyroglobulin synthesis and cytoplasmic mRNA content to undetectable levels. Addition of TSH to cells previously exposed to EGF reversed the growth factor effect on all four indexes. The redifferentiating effect of TSH was well observed within 3-4 days and was mimicked by the adenylate cyclase activators, forskolin and cholera toxin. When administered simultaneously, TSH and EGF achieved an intermediate situation, EGF antagonizing partially the effect of TSH on the expression of thyroglobulin gene. Another growth factor, fibroblast growth factor, while promoting thyroid cell proliferation also, did not interfere at all with TSH effects on cytoplasmic thyroglobulin mRNA content. Our results make the dog thyroid cell in primary culture an appropriate model to study the mechanisms involved in gene regulation by cyclic AMP and growth factors.     

Clonal variations in keratin : Intermediate filament expression by human somatic cell hybrids

The intermediate filament composition of differentiated vertebrate cells provides a stable phenotype which appears to be specifically regulated in each cell type. In order to analyse the regulation of intermediate filament expression we have constructed human somatic cell hybrids from the fusion of the HeLa-derived cell line HEB7A and a normal human diploid fibroblast, GM2291. These parental cells differ with respect to the presence or absence of keratin intermediate filaments. Isolation of independently arising clones produced two classes of hybrids. One class expresses keratin in a stable manner and the other class lacks keratin altogether. Indirect immunofluorescence of hybrid cells using antikeratin antiserum demonstrates that there are variations in the intensity and organization of cytoskeletal keratin staining. SDS-PAGE comparisons of cell extracts from these hybrids indicates that there are quantitative differences in the relative amounts of individual keratin polypeptides as well. These clonal variations have allowed us to begin assessing the consequences of genetic interactions between cell types that are normally capable of closely regulating different subsets of intermediate filament genes.     

The distribution and temporal dynamics of the estuarine macroalgal community of San Francisco Bay

Long-term sampling of intertidal macroalgae along permanently marked transects within San Francisco Bay has shown a marked decline in overall species number along the estuarine gradient from the ocean to the river, presumably as a result of decreasing salinity and a progressive lack of hard substrata in the upstream direction. Green algae penetrated further landward than either brown or red species. Seasonally, macroalgal species diversity is lowest during the winter-spring months when salinity, temperature, and irradiance are at yearly minima. Macroalgal abundance as measured by percent cover was maximum during the late spring near the mouth of the estuary and during late summer towards the head. The seasonal increase in algal abundance is related to increasing salinity, temperature, and light availability to the bottom. The summer increase in irradiance is due to the longer photoperiod, increased frequency of day-time low tides, and reduced levels of suspended sediments. The aperiodic occurrence of algal blooms in San Pablo Bay may be caused by a combination of physical factors which are ultimately associated with the river inflow. A hypothesis based on interannual differences in river inflow and the contribution of phytoplankton to nutrient cycles in the benthos is presented to explain the occurrence of nuisance algal blooms.     

Method for gene replacement in Pseudomonas aeruginosa used in construction of recA mutants: recA-independent instability of alginate production.

The availability of a technique for site-directed mutagenesis by gene replacement provides a powerful tool for genetic analysis in any bacterial species. We report here a general technique for gene replacement in Pseudomonas aeruginosa. Genes on fragments of cloned P. aeruginosa DNA, altered by transposon mutagenesis, can be transduced into a recipient strain and can replace homologous genes in the P. aeruginosa genome. In this study we applied this technique to the construction of recA mutants of P. aeruginosa. A cloned segment of P. aeruginosa FRD1 DNA was isolated which encoded a protein analogous to the recA gene product of Escherichia coli. The P. aeruginosa recA gene was able to complement several defects associated with recA mutation in E. coli. Transposon Tn1 and Tn501 insertions in the cloned recA gene of P. aeruginosa were used to generate chromosomal recA mutants by gene replacement. These recA strains of P. aeruginosa were more sensitive to UV irradiation and methyl methane sulfonate and showed reduced recombination proficiency compared with the wild type. Also examined was the effect of recA mutations on the expression of alginate, a virulence trait. Alginate is a capsulelike polysaccharide associated with certain pulmonary infections, and its expression is typically unstable. The genetic mechanism responsible for the instability of alginate biosynthesis was shown to be recA independent.     

Partial Purification and Properties of Human Brain Aldehyde Dehydrogenases

Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.