Mendelian inheritance in Germany between 1900 and 1910. The case of Carl Correns (1864-1933).

Carl Correns (1864-1933) came to recognize Mendel's rules between 1894 and 1900 while trying to find out the mechanism of xenia, that is, the direct influence of the fertilizing pollen on the mother plant in maize and peas among other species. In this paper, I am concerned with the ten years of Correns' work after the annus mirabilis of 1900 until 1910, when the main outlines of the new science of genetics had been established. It is generally assumed that after 1900 Correns quickly began probing the limits of Mendelian inheritance, both as far as the explanatory force of formal transmission genetics and the generality of Mendel's laws are concerned. A careful examination of his papers however shows that he was much more interested in the scope of Mendelian inheritance than in its limits. Even his work with variegated Mirabilis plants, which historiographical folklore still presents as a result of Correns' growing interest in cytoplasmic inheritance, can be shown to have been conducted to corroborate just the opposite, namely, the validity of the nuclear paradigm. The paper will show that Correns' research results in those years (among them the Mendelian inheritance of sex in higher plants) were the outcome of a complex experimental program which involved breeding experiments with dozens of different species.

Ephestia: the experimental design of Alfred Kühn's physiological developmental genetics.

Much of the early history of developmental and physiological genetics in Germany remains to be written. Together with Carl Correns and Richard Goldschmidt, Alfred Kühn occupies a special place in this history. Trained as a zoologist in Freiburg im Breisgau, he set out to integrate physiology, development and genetics in a particular experimental system based on the flour moth Ephestia kühniella Zeller. This paper is meant to reconstruct the crucial steps in the experimental pathway that led Kühn and his collaborators at the University of Göttingen, and later at the Kaiser Wilhelm Institutes of Biology and Biochemistry in Berlin, to formulate, in their specific way, what later became known as the "one gene - one enzyme hypothesis." Special attention will be given to the interaction of the different parts of Kühn's Ephestia-based project, which were rooted in different research traditions. The paper retraces how, roughly between 1925 and 1945, these elements came to form a mixed experimental set-up composed of genetic, embryological, physiological and, finally, biochemical constituents. Accordingly, emphasis is laid on the development of the terminology in which the results were cast, and how it reflected the hybrid state of an experimental system successively acquiring new epistemic layers.

[Carl Correns' experiments with Pisum, 1896-1899]. / Carl Correns' Experimente mit Pisum, 1896-1899.

The circumstances under which classical genetics became established at the turn of the nineteenth century have become an integral part of the standard narrative on the history of genetics. Yet, despite considerable scholarly efforts, it has remained a matter of debate how exactly the so-called 'rediscovery' of Mendel's laws came about around 1900. In this situation, unpublished research records can be invaluable tools to arrive at a more substantial and more satisfying picture of the order of historical events. This paper makes extended use of the research protocols covering Carl Correns' hybridisation experiments with Pisum sativum between 1896 and 1899. The resulting reconstruction sketches the portrait of a scientist following a particular research question--xenia--struggling with his experimental material, and slowly building up an epistemic regime in which questions and observations could acquire a relevance which did not strike Correns when he first took note of them. The microhistorical gaze through the magnifying glass of research notes reveals the kind of delays that appear to be constitutive for empirically-driven thinking in general. The research notes of Correns help not only to make this point, they also display some of the intricacies and material peculiarities which characterise the experimental process of hybridisation and the particular type of inferences it allows one to make.

Modulation of IgE reactivity of allergens by site-directed mutagenesis: potential use of hypoallergenic variants for immunotherapy.

Specific immunotherapy is an efficient treatment for patients suffering from type I allergy. The mechanisms underlying successful immunotherapy are assumed to operate at the level of T helper cells, leading to a modulation of the immune response to allergens. During immunotherapy, increasing doses of allergens are given on a regular basis, and the beneficial effects for the patient depend on the concentration of allergen used. On the other hand, the risk of IgE-mediated anaphylactic side effects also increase with the amount of allergen applied per injection. Therefore, we have proposed the use of hypoallergenic (low IgE binding activity) forms of allergens for immunotherapy. We evaluated by site-directed mutagenesis the contributions of individual amino acid residues/positions for IgE binding to Bet v 1, the major allergen of birch pollen. We found that IgE binding to Bet v 1 depended on at least six amino acid residues/positions. Immunoblot analyses and inhibition experiments showed that the multiple-point Bet v 1 mutant exhibited extremely low reactivity with serum IgE from birch pollen-allergic patients. In vivo (skin prick) tests showed that the potency of the multiple-point mutant to induce typical urticarial type I reactions in pollen-allergic patients was significantly lower than for wild-type Bet v 1. Proliferation assays of allergen-specific T cell clones demonstrated that these six amino acid exchanges in the Bet v 1 sequence did not influence T cell recognition. Thus, the Bet v 1 six-point mutant displayed significantly reduced IgE binding activity, but conserved T cell activating capacity, which is necessary for immunomodulation. The approach described here may be generally applied to produce allergen variants to be used in a safe therapy form of immediate-type allergies.

Immunological and biological properties of Bet v 4, a novel birch pollen allergen with two EF-hand calcium-binding domains.

We have isolated a cDNA clone coding for a birch pollen allergen, Bet v 4. The deduced amino acid sequence of Bet v 4 contained two typical EF-hand calcium-binding domains. Sequence similarities of Bet v 4 to calmodulin are primarily confined to the calcium-binding domains. However, significant sequence similarities extending outside the Ca2+-binding sites were found with a recently described group of pollen-specific allergens of Brassica and Bermuda grass. Both EF-hand domains of Bet v 4 are able to bind Ca2+, as demonstrated by 45Ca2+ blot overlay of wild type and calcium-binding deficient mutants of Bet v 4. Among pollen-allergic patients, protein-bound Ca2+ was not an absolute requirement for IgE recognition of Bet v 4. However, disruption of the carboxyl-terminal Ca2+-binding domain indicated that most IgE antibodies from allergic patients are directed against this site. IgE inhibition experiments suggested that Bet v 4 represents a highly cross-reactive pollen allergen. Pre-absorption of allergic sera with Bet v 4 drastically reduced IgE binding to proteins of similar molecular weight in pollen extracts from distantly related plant species (e.g. timothy grass, mugwort, lily) but not in extracts from plant-derived foodstuff. To test for a possible biological role in pollen germination and tube growth, we introduced recombinant Bet v 4 protein into growing lily pollen tubes by iontophoresis. As a result, cytoplasmic streaming stopped in the vicinity of the electrode tip, and a slight depolarization of the membrane voltage was measured. These effects were not observed with Ca2+-binding deficient mutants of Bet v 4. Thus, Bet v 4 and homologous proteins represent a new class of pollen-specific Ca2+-binding allergens that may have a physiological role as inhibitors of cytoplasmic streaming in outgrowing pollen tubes.

Dissection of immunoglobulin E and T lymphocyte reactivity of isoforms of the major birch pollen allergen Bet v 1: potential use of hypoallergenic isoforms for immunotherapy.

We dissected the T cell activation potency and the immunoglobulin (Ig) E-binding properties (allergenicity) of nine isoforms of Bet v 1 (Bet v 1a-Bet v 1l), the major birch pollen allergen. Immunoblot experiments showed that Bet v 1 isoforms differ in their ability to bind IgE from birch pollen-allergic patients. All patients tested displayed similar IgE-binding patterns toward each particular isoform. Based on these experiments, we grouped Bet v 1 isoforms in three classes: molecules with high IgE-binding activity (isoforms a, e, and j), intermediate IgE-binding (isoforms b, c, and f), and low/no IgE-binding activity (isoforms d, g, and 1). Bet v 1a, a recombinant isoform selected from a cDNA expression library using IgE immunoscreening exhibited the highest IgE-binding activity. Isoforms a, b, d, e, and 1 were chosen as representatives from the three classes for experimentation. The potency of each isoallergen to activate T lymphocytes from birch pollen-allergic patients was assayed using peripheral blood mononuclear cells, allergen-specific T cell lines, and peptide-mapped allergen-specific T cell clones. Among the patients, some displayed a broad range of T cell-recognition patterns for Bet v 1 isoforms whereas others seemed to be restricted to particular isoforms. In spite of this variability, the highest scores for T cell proliferative responses were observed with isoform d (low IgE binder), followed by b, 1, e, and a. In vivo (skin prick) tests showed that the potency of isoforms d and 1 to induce typical urticarial type 1 reactions in Bet v 1-allergic individuals was significantly lower than for isoforms a, b, and e. Taken together, our results indicate that hypoallergenic Bet v 1 isoforms are potent activators of allergen-specific T lymphocytes, and Bet v 1 isoforms with high in vitro IgE-binding activity and in vivo allergenicity can display low T cell antigenicity. Based on these findings, we propose a novel approach for immunotherapy of type I allergies: a treatment with high doses of hypoallergenic isoforms or recombinant variants of atopic allergens. We proceed on the assumption that this measure would modulate the quality of the T helper cell response to allergens in vivo. The therapy form would additionally implicate a reduced risk of anaphylactic side effects.

Beyond nature and culture: a note on medicine in the age of molecular biology.

This paper is divided into two parts. In the first, I examine the relations among molecular biology, gene technology, and medicine, as well as some aspects of the consequences of these relations with respect to the human genome project. I argue that the prevailing momentum of early molecular biology resided in creating the technical means for an extracellular representation of intracellular configurations. As such, its medical impact was rather limited. With the advent of recombinant DNA technologies, a radical change of perspective ensued. The momentum of gene technology is based on the prospects of an intracellular representation of extracellular projects--the "rewriting" of life. Its medical impact is potentially unlimited. In the second part, I question the very opposition between nature and culture that implicitly underlies the notion of medicine as a "cultural system." I argue that both on a macroscopic level (global ecological changes) and on a microscopic level (genetic engineering), the "natural" and the "social" are no longer to be seen as ontologically different. In its uncanny oscillation between retrospection and foresight, between description and proclamation, and between assertion and hesitatiion, this essay translates an uneasiness that I have not been able to overcome while writing it. The essay conveys the tangled views of a hybrid author who himself cannot but oscillate between the perspectives of an actor in the field of molecular biology, a participant in the field of science studies, and a citizen.

The function of the translating ribosome: allosteric three-site model of elongation.

During the last decade, a new model for the ribosomal elongation cycle has emerged. It is based on the finding that eubacterial ribosomes possess 3 tRNA binding sites. More recently, this has been confirmed for archaebacterial and eukaryotic ribosomes as well, and thus appears to be a universal feature of the protein synthetic machinery. Ribosomes from organisms of all 3 kingdoms harbor, in addition to the classical P and A sites, an E site (E for exit), into which deacylated tRNA is displaced during translocation, and from which it is expelled by the binding of an aminoacyl-tRNA to the A site at the beginning of the subsequent elongation round. The main features of the allosteric 3-site model of ribosomal elongation are the following: first, the third tRNA binding site is located 'upstream' adjacent to the P site with respect to the messenger, ie on the 5'-side of the P site. Second, during translocation, deacylated tRNA does not leave the ribosome from the P site, but co-translocates from the P site to the E site--when peptidyl-tRNA translocates from the A site to the P site. Third, deacylated tRNA is tightly bound to the E site in the post-translocational state, where it undergoes codon--anticodon interaction. Fourth, the elongating ribosome oscillates between 2 main conformations: (i), the pre-translocational conformer, where aminoacyl-tRNA (or peptidyl-tRNA) and peptidyl-tRNA (or deacylated tRNA) are firmly bound to the A and P sites, respectively; and (ii), the post-translocational conformer, where peptidyl-tRNA and deacylated tRNA are firmly bound to the P and E sites, respectively. The transition between the 2 states is regulated in an allosteric manner via negative cooperatively. It is modulated in a symmetrical fashion by the 2 elongation factors Tu and G. An elongating ribosome always maintains 2 high-affinity tRNA binding sites with 2 adjacent codon--anticodon interactions. The allosteric transition from the post- to the pre-translocational state is involved in the accuracy of aminoacyl-tRNA selection, and the maintenance of 2 codon--anticodon interactions helps to keep the messenger in frame during translation.

Partial release of AcPhe-Phe-tRNA from ribosomes during poly(U)-dependent poly(Phe) synthesis and the effects of chloramphenicol.

Poly(U)-programmed 70S ribosomes can be shown to be 80% to 100% active in binding the peptidyl-tRNA analogue AcPhe-tRNA to their A or P sites, respectively. Despite this fact, only a fraction of such ribosomes primed with AcPhe-tRNA participate in poly(U)-directed poly(Phe) synthesis (up to 65%) at 14 mM Mg2+ and 160 mM NH4+. Here it is demonstrated that the apparently 'inactive' ribosomes (greater than or equal to 35%) are able to participate in peptide-bond formation, but lose their nascent peptidyl-tRNA at the stage of Ac(Phe)n-tRNA, with n greater than or equal to 2. The relative loss of early peptidyl-tRNAs is largely independent of the degree of initial saturation with AcPhe-tRNA and is observed in a poly(A) system as well. This observation resolves a current controversy concerning the active fraction of ribosomes. The loss of Ac(Phe)n-tRNA is reduced but still significant if more physiological conditions for Ac(Phe)n synthesis are applied (3 mM Mg2+, 150 mM NH4+, 2 mM spermidine, 0.05 mM spermine). Chloramphenicol (0.1 mM) blocks the puromycin reaction with AcPhe-tRNA as expected but, surprisingly, does not affect the puromycin reaction with Ac(Phe)2-tRNA nor peptide bond formation between AcPhe-tRNA and Phe-tRNA. The drug facilitates the release of Ac(Phe)2-4-tRNA from ribosomes at 14 mM Mg2+ while it hardly affects the overall synthesis of poly(Phe) or poly(Lys).

[Buffon: time, change and history]. / Buffon: Zeit, Veränderung und Geschichte.

There is a longstanding and ongoing controversy about whether Buffon is to be regarded as a forerunner of evolutionism in the eighteenth century, or even as one of the founders of transformistic biology. There are good reasons to deny this claim. There are good reasons even to deny that the question which is going to be answered negatively is of particular importance. The present paper addresses the issue from a different angle. It analyzes the concept of time operative in the natural history writings of Buffon, and it delineates the articulation of the concepts of time, change, and history with its organizing impact on Buffon's discourse on earth and organisms. It is argued that although with his species concept Buffon tries to introduce the classical notion of a physical system into biology, in order to do so, he has to subvert it by an element of time. This guides him in considering various aspects of organic change, but by itself does not lead to a general perspective of transformation. On the other hand, in his Epoques de la nature, Buffon introduces a general law of geological change, thus arriving at something which could be called a physically intelligible history. The conquest of natural history by physics, in one and the same movement, leads to a subversion of physical geology by history, and prevents biology from becoming evolutionistic in the sense in which the nineteenth century understands this term.

The allosteric three-site model for the ribosomal elongation cycle. Analysis with a heteropolymeric mRNA.

Ribosomal tRNA binding studies and functional tests were performed at 6 mM Mg2+ using the mRNA analogue C17AUGA4C17 which contains three unique codons in its central region. The following results were obtained. 1) The relative binding affinities of 20 different deacylated tRNAs to nonprogrammed 70 S ribosomes were assessed and were found to vary substantially. 2) When added as the first tRNA, fMet-tRNA and deacylated tRNAs (but not N-acetylated aminoacyl-tRNAs) can bind to internal codons of the mRNA and are therefore suitable for setting the reading frame via codon-anticodon interaction in the peptidyl-tRNA site (P site). 3) After prefilling the P site with deacylated tRNA, the exit site for deacylated tRNA (E site) can be quantitatively occupied only if the cognate codon is present at that site. 4) The translocation of peptidyl-tRNA from the aminoacyl-tRNA site (A site) to the P site is not accompanied by a release of deacylated tRNA. The codon sequence excludes a release and rebinding of deacylated tRNA to the newly exposed A site. Rather, the deacylated tRNA is cotranslocated from the P to the E site where it remains stably bound. 5) After one round of elongation, addition of an A site ligand triggers the dissociation of deacylated tRNA from the E site. Conversely, E site occupation reduces the affinity of the A site for N-acetylated aminoacyl-tRNA. Thus, A and E sites are allosterically linked via negative cooperativity. The results support the allosteric three-site model as an appropriate description of the ribosomal elongation cycle.