Characterization of the Late Steps of Microbial Heme Synthesis: Conversion of Coproporphyrinogen to Protoporphyrin

Cell-free extracts of various cytochrome-containing, heterotrophic microorganisms were examined for ability to convert coproporphyrinogen to protoporphyrin. Extracts of Escherichia coli and Pseudomonas denitrificans readily accumulated large amounts of protoporphyrin when assayed under aerobic conditions. However, protoporphyrin did not accumulate under either aerobic or anaerobic conditions of assay or in the presence of various supplements in extracts of the aerobe Micrococcus lysodeikticus, the facultative anaerobe Staphylococcus aureus, or the anaerobe Vibrio succinogenes. Protoporphyrin also accumulated when extracts of E. coli and P. denitrificans were incubated aerobically with the early heme precursor, delta-amino levulinic acid (ALA). This protoporphyrin accumulation was markedly stimulated by the iron chelator, o-phenanthroline. Extracts of S. aureus and M. lysodeikticus accumulated coproporphyrin, but not protoporphyrin when incubated with ALA. The enzyme system in extracts of E. coli which converts coproporphyrinogen to protoporphyrin under aerobic conditions of assay was also partially characterized. This conversion was stimulated by the iron chelator, o-phenanthroline, the respiratory inhibitor, cyanide, and the reducing agent, thioglycolate. Dialysis of the extract did not diminish enzyme activity. Certain alternate electron acceptors and nitrite caused a marked inhibition of the conversion. These results indicate that this late step in heme synthesis, the conversion of coproporphyrinogen to protoporphyrin, can be readily demonstrated in extracts of some, but not all, cytochrome-containing bacteria and that the aerobic conversion in E. coli exhibits many characteristics similar to those demonstrated for the aerobic conversion previously studied in liver mitochondria.     

Structural Analysis of the dur Loci in S. CEREVISIAE: Two Domains of a Single Multifunctional Gene

In Saccharomyces cerevisiae, the degradation of urea to carbon dioxide and ammonia is catalyzed by urea carboxylase and allophanate hydrolase. The loci coding for these enzymes (dur1 and dur2) are very tightly linked on the right arm of chromosome II between pet11 and met8. Pleiotropic mutations that fail to complement mutations in either of the dur loci were found to be predominantly located in or near the dur2 locus. We interpret these data as suggesting that the two dur loci might in reality be domains of a single gene that codes for a multifunctional polypeptide. In view of this conclusion, we have renamed the dur loci as the dur1,2 locus.     

Gene Duplication within the Family Salmonidae: Disomic Inheritance of Two Loci Reported to Be Tetrasomic in Rainbow Trout

We describe our studies of the genetics of allelic variation for NADP-dependent isocitrate dehydrogenase (IDH) in rainbow trout (Salmo gairdneri). Five populations of rainbow trout were studied to determine the phenotypic distribution of IDH; 453 progeny from a number of controlled matings were examined to determine the nature of inheritance of these alleles. The variation was found to be the result of four alleles producing protein subunits of differing electrophoretic mobilities. Progeny from crosses clearly demonstrated the presence of two disomic loci controlling the variation, rather than one tetrasomic locus as had been previously reported. These findings support our contention that the hypothesis of a tetraploid event in salmonid evolution should not be uncritically accepted.     

FLAGELLAR ELONGATION AND SHORTENING IN CHLAMYDOMONAS : The Use of Cycloheximide and Colchicine to Study the Synthesis and Assembly of Flagellar Proteins

Flagella can be removed from the biflagellate Chlamydomonas and the cells begin to regenerate flagella almost immediately by deceleratory kinetics. Under usual conditions of deflagellation, more than 98% of all flagella are removed. Under less drastic conditions, cells can be selected in which one flagellum is removed and the other left intact. When only one of the two flagella is amputated, the intact flagellum shortens by linear kinetics while the amputated one regenerates. The two flagella attain an equal intermediate length and then approach their initial length at the same rate. A concentration of cycloheximide which inhibits protein synthesis permits less than one-third of each flagellum to form when both flagella are amputated. When only one is amputated in cycloheximide, shortening proceeds normally and the degree of elongation in the amputated flagellum is greater than if both were amputated in the presence of cycloheximide. The shortening process is therefore independent of protein synthesis, and the protein from the shortening flagellum probably enters the pool of precursors available for flagellar formation. Partial regeneration of flagella occurs in concentrations of cycloheximide inhibitory to protein synthesis suggesting that some flagellar precursors are present. Cycloheximide and flagellar pulse-labeling studies indicate that precursor is used during the first part of elongation, is resynthesized at mid-elongation, and approaches its original level as the flagella reach their initial length. Colchicine completely blocks regeneration without affecting protein synthesis, and extended exposure of deflagellated cells to colchicine increases the amount of flagellar growth upon transfer to cycloheximide. When colchicine is applied to cells with only one flagellum removed, shortening continues normally but regeneration is blocked. Therefore, colchicine can be used to separate the processes of shortening and elongation. Radioautographic studies of the growth zone of Chlamydomonas flagella corroborate previous findings that assembly is occurring at the distal end (tip growth) of the organelle.     

Marvelling at the Marvel: The Supposed Conversion of A.D. Darbishire to Mendelism

The so-called “biometric-Mendelian controversy” has received much attention from science studies scholars. This paper focuses on one scientist involved in this debate, Arthur Dukinfield Darbishire, who performed a series of hybridization experiments with mice beginning in 1901. Previous historical work on Darbishire’s experiments and his later attempt to reconcile Mendelian and biometric views describe Darbishire as eventually being “converted”' to Mendelism. I provide a new analysis of this episode in the context of Darbishire’s experimental results, his underlying epistemology, and his influence on the broader debate surrounding the rediscovery and acceptance of Mendelism. Iinvestigate various historiographical issues raised by this episode in order to reflect on the idea of “conversion” to a scientific theory. Darbishire was an influential figure who resisted strong forces compelling him to convert prematurely due to his requirements that the new theory account for particularly important anomalous facts and answer the most pressing questions in the field. This revised version was published online in July 2006 with corrections to the Cover Date.     

Viability of Chlamydomonas Mutants with Amino Acid Substitutions in the Precursor D1 Protein at the Carboxyl-Terminal Processing Site: an Analysis by Mixed-Culture Growth Experiments

Department of Biology, Faculty of Science, Okayama University,3-1-1, Tsushima-naka, Okayama, 700-8530 Japan In order to analyzethe influence of amino acid substitutions at the carboxyl-terminalprocessing site of the D1 precursor protein, mixed-culture growthexperiments were conducted for psbA directed mutants of Chlamydomonasreinhardtii. Wild type and D1 mutants were mixed in the sameculture and their viability was compared. Replacement of Ser-345by Gly or Val at the cleavage site markedly affected the relativegrowth rate of the mutant in the high intensity light, but notin a dim light or the darkness. This was consistent with theprevious result obtained by in vitro analysis using substitutedcarboxyl-terminal oligopeptides as substrates [Taguchi et al.(1995) J Biol. Chem. 270: 10711], This is a clear indicationthat the rate of carboxyl-terminal processing of the D1 precursorin the photosystem II reaction center is a rate-limiting stepfor growth under some environmental stress conditions. (Received June 9, 1998; Accepted September 25, 1998)     

Gene Conversion at the Gray Locus of SORDARIA FIMICOLA: Fit of the Experimental Data to a Hybrid DNA Model of Recombination

A hybrid DNA (hDNA) model of recombination has been algebraically formulated, which allows the prediction of frequencies of postmeiotic segregation and conversion of a given allele and their probability of being associated with a crossing over. The model considered is essentially the "Aviemore model." In contrast to some other interpretations of recombination, it states that gene conversion can only result from the repair of heteroduplex hDNA, with postmeiotic segregation resulting from unrepaired heteroduplexes. The model also postulates that crossing over always occurs distally to the initiation site of the hDNA. Eleven types of conversion and postmeiotic segregation with or without associated crossover were considered. Their theoretical frequencies are given by 11 linear equations with ten variables, four describing heteroduplex repair, four giving the probability of hDNA formation and its topological properties and two giving the probability that crossing over occurs at the left or right of the converting allele. Using the experimental data of Kitani and coworkers on conversion at the six best studied gray alleles of Sordaria fimicola, we found that the model considered fit the data at a P level above or very close (allele h4) to the 5% level of sampling error provided that the hDNA is partly asymmetric. The best fitting solutions are such that the hDNA has an equal probability of being formed on either chromatid or, alternatively, that both DNA strands have the same probability of acting as the invading strand during hDNA formation. The two mismatches corresponding to a given allele are repaired with different efficiencies. Optimal solutions are found if one allows for repair to be more efficient on the asymmetric hDNA than on the symmetric one. In the case of allele g1, our data imply that the direction of repair is nonrandom with respect to the strand on which it occurs.     

ORIGIN OF GRANULES IN POLYMORPHONUCLEAR LEUKOCYTES : Two Types Derived from Opposite Faces of the Golgi Complex in Developing Granulocytes

The origin, nature, and distribution of polymorphonuclear leukocyte (PMN) granules were investigated by examining developing granulocytes from normal rabbit bone marrow which had been fixed in glutaraldehyde and postfixed in OsO₄. Two distinct types of granules, azurophil and specific, were distinguished on the basis of their differences in size, density, and time and mode of origin. Both types are produced by the Golgi complex, but they are formed at different stages of maturation and originate from different faces of the Golgi complex. Azurophil granules are larger (~800 mµ) and more dense. They are formed only during the progranulocyte stage and arise from the proximal or concave face of the Golgi complex by budding and subsequent aggregation of vacuoles with a dense core. Smaller (~500 mµ), less dense specific granules are formed during the myelocyte stage; they arise from the distal or convex face of the Golgi complex by pinching-off and confluence of vesicles which have a finely granular content. Only azurophil granules are found in progranulocytes, but in mature PMN relatively few (10 to 20%) azurophils are seen and most (80 to 90%) of the granules present are of the specific type. The results indicate that inversion of the azurophil/specific granule ratio occurs during the myelocyte stage and is due to: (a) reduction of azurophil granules by multiple mitoses; (b) lack of new azurophil granule formation after the progranulocyte stage; and (c) continuing specific granule production. The findings demonstrate the existence of two distinct granule types in normal rabbit PMN and their separate origins from the Golgi complex. The implications of the observations are discussed in relationship to previous morphological and cytochemical studies on PMN granules and to such questions as the source of primary lysosomes and the concept of polarity within the Golgi complex.     

Two alternatives to the stress‐gradient hypothesis at the edge of life: the collapse of facilitation and the switch from facilitation to competition

New evidence demonstrates that facilitation plays a crucial role even at the edge of life in Maritime Antarctica. These findings are interpreted as support for the stress‐gradient hypothesis (SGH) – a dominant theory in plant community ecology that predicts that the frequency of facilitation directly increases with stress. A recent development to this theory, however, proposed that facilitation often collapses at the extreme end of stress and physical disturbance gradients. In this paper, we clarify the current debate on the importance of plant interactions at the edge of life by illustrating the necessity of separating the two alternatives to the SGH, namely the collapse of facilitation, and the switch from facilitation to competition occurring in water‐stressed ecosystems. These two different alternatives to the SGH are currently often amalgamated with each other, which has led to confusion in recent literature. We propose that the collapse of facilitation is generally due to a decrease in the effect of the nurse plant species, whilst the switch from facilitation to competition is driven by environmental conditions and strategy of the response species. A clear separation between those two alternatives is particularly crucial for predicting the role of plant–plant interactions in mediating species responses to global change.     

Conversion of the FokI endonuclease to a universal restriction enzyme: cleavage of phage M13mp7 DNA at predetermined sites

Endonuclease FokI belongs to class IIS of restriction enzymes, for which the DNA cut points lie outside the enzyme-recognition sites. This permitted conferring new cleavage specificities by combining FokI with tailored oligodeoxynucleotide adapters. Such adapters carry a single-stranded (ss) target-recognition domain, complementary to the selected ss target DNA, and a double-stranded (ds) enzyme-recognition site. Neither enzyme nor adapter alone has endonucleolytic activity toward phage M13mp7 ss DNA, whereas the enzymeadapter complex cleaves this ss target DNA at the particular sites foreordained by the sequence of the ss domain of the adapter. Two kinds of adapters (32 and 34 nucleotides long), with opposing orientations of the asymmetric FokI recognition site, were constructed and shown to direct specific cleavage under a variety of conditions. In addition to FokI, other class IIS enzymes, HphI, MboII and BbvI, which alone do not cleave ss DNA, are suitable for construction of tailored enzyme-adapter complexes with predictable cleavage specificities.

This report provides a preliminary experimental confirmation for the proposal of Szybalski [Gene 40 (1985) 169-173] for the design of adapter-enzyme complexes with novel and predictable specificities. Theoretically, using this approach any sequence could be precisely cleaved at a predetermined point.     

On Analysis of the Difference of Two Exposure-Adjusted Poisson Rates with Stratification: From Asymptotic to Exact Approaches

Long-term studies are frequently conducted to assess the treatment effect on rare diseases or the safety of a new treatment. To account for differential follow-up often encountered in long-term studies, exposure-adjusted incidence rates are used in evaluating the treatment effect. The difference of rates is sometimes used to quantify the treatment’s public health impact because the reciprocal of this difference can be interpreted as “the number needed to treat (or number needed to vaccinate) in order to cure (or prevent) 1 case of disease.” In this paper we focus on the stratified analysis of the difference of two exposure-adjusted rates in the setting of superiority, noninferiority and super-superiority hypothesis testing. After a brief review of asymptotic methods, we derive an exact method that guarantees control of the type I error. But it is conservative for noninferiority and super-superiority testing because of the search of the maximum tail probability over a multidimensional nuisance parameter space. Then, we present an approximate exact method where the p-value is estimated at the maximum likelihood estimates of the nuisance parameter. This method is identical to exact method for superiority testing and reduces the conservatism for noninferiority and super-superiority testing. In addition, a quasi-exact method is discussed. A real-life vaccine clinical trial example is used to illustrate these methods. Finally, we compare the performance of these methods via empirical studies and make some general practical recommendations.     

Analysis of DNA damage at the dinucleoside monophosphate level: application to the formamido lesion.

The dinucleoside monophosphates d(TpG), d(TpC), and d(TpT) were X-irradiated in oxygenated solution. In each case the modification of the dinucleoside in which the thymine base is degraded to a formamido remnant was observed as a principal product. The hydrolysis of the phosphoester bond of formamido-modified dinucleosides is much slower than that of the corresponding unmodified dinucleosides. This effect is also observable in the hydrolysis of irradiated DNA, where hydrolysis by nuclease P1 (plus acid phosphatase) generates the modified dinucleosides d(TFpN), TF being the modified thymidine. The total yield of the formamido lesion in all its forms, d(TFpN), exceeds the yield of any other base modification.