Genetics of susceptibility to tuberculosis: Mengele's experiments in Auschwitz.

Great experiments will always be remembered. I highlight an experiment that was conducted during the Nazi regime in Germany. Not only did the experiment fail, it was also linked to fraud and crimes against humanity. This failed experiment will never be forgotten.

High local protein concentrations at promoters: strategies in prokaryotic and eukaryotic cells.

The speed of chemical reactions is proportional to the concentration of molecules involved. Since proteins catalyze most of the essential reactions inside a living cell, their concentration should be as high as possible. An economical way to achieve this is through the establishment of small cell compartments. We propose that within these compartments, two types of local concentration effects are at work. (1) With local concentration type I reactions, multimeric proteins bound to a specific DNA sequence have an increased local concentration for a second DNA site sufficiently close-by, or for proteins bound to such a site. (2) For type II effects, DNA can be used as a scaffold to build unique nucleoprotein complexes that would otherwise not exist free in solution. These complexes are proficient in establishing longer-range interactions with similarly unique complexes located far away on the genome. We discuss the consequences of these local concentration effects in the light of the markedly different sizes of prokaryotic and eukaryotic cells and of their genomes.

Reflections on the use of interviews in the history of science: who will write the history of science?

Interviews are an excellent source of information for historians of science. They should be done by historians who understand science in detail and, if possible, better than the scientists they interview. In the case of applied industrial or governmental sciences, historians must have detailed knowledge of economic or historic sources. Again they should know more in these areas than those they interview. If, on the contrary, the interviewers are not scientists at heart who know science, the history they write will become at best literature but at worst pseudoscientific abracadabra.

On the conservation of protein sequences in evolution.

The amino acid sequences of enzymes like alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase are strongly conserved across all phyla. We suggest that the amino acid conservation of such enzymes might be a result of the fact that they function as part of a multi-enzyme complex. The specific interactions between the proteins involved would hinder evolutionary change of their surfaces.

Strengthening the dimerisation interface of Lac repressor increases its thermostability by 40 deg. C.

We increased drastically the heat stability of Lac repressor (LacR) of Escherichia coli. Wild-type tetrameric LacR denatures irreversibly at 53 degrees C. Improving hydrophobic packing at the dimerisation interface by a single substitution increases LacR heat-resistance by 40 deg. C without abolishing inducer binding at high and low temperatures. Tetrameric LacR mutants carrying substitutions of the positively charged amino acid Lys84 by each of the hydrophobic amino acids Leu, Ile and Met resist heating to temperatures up to 93 degrees C. We performed IPTG binding assays at 80 degrees C and found the mutant Lac repressors active and, thus, the core intact. Furthermore, the activity of LacR following heating is shown at room temperature by a gel retardation assay, which demonstrates normal oligomerisation state and function of the headpiece. The same mutations (K84L/I/M) in the dimer LacR331stop, carrying a stop codon in amino acid 331, increase thermostability of the dimer from 47 degrees C to 87 degrees C. LacRK84M represses beta-galactosidase activity in vivo as well as the wild-type and is sufficiently induced to allow growth on lactose. The results with both tetramer and dimer variants of LacR indicate mutual stabilisation of the tetramerisation region and the stable core.

Dimerisation mutants of Lac repressor. II. A single amino acid substitution, D278L, changes the specificity of dimerisation.

Assembly of the lactose repressor tetramer involves two subunit interfaces, the C-terminal heptad repeats, and the monomer-monomer interface. Dimerisation between two monomers of Lac repressor of Escherichia coli lacking the two C-terminal heptad repeats occurs through the interactions between three alpha-helices of each monomer, which form a highly hydrophobic interface. Residues possibly involved in specific dimer formation are known from X-ray studies and from the phenotypes of more than 4000 single amino acid substitutions. During the examination of numerous mutants within the dimerisation interface of Lac repressor, we found that substitution of one amino acid, D278 to leucine, is sufficient to change the specificity of dimerisation. Analysis of this single substitution indicates that D278L mutant Lac repressor represses like wild-type. However, it no longer forms heterodimers with wild-type Lac repressor.

Dimerisation mutants of Lac repressor. I. A monomeric mutant, L251A, that binds Lac operator DNA as a dimer.

Dimer formation between monomers of the Escherichia coli Lac repressor is substantially specificed by the interactions between three alpha-helices in each monomer which form a hydrophobic interface. As a first step in analysing the specificity of this interaction, we examined the mutant L251A. LacR bearing this mutation in a background lacking the C-terminal heptad repeats is completely incapable of forming dimers in solution, with a dimer-monomer equilibrium dissociation constant, or Kd, higher than 10(-5)M. This correlates with a 200-fold decrease in its ability to repress the lac operon in vivo compared to dimeric LacR. Surprisingly, the mutant is still capable of forming dimers upon binding to short operator DNA in vitro. Analysis of the kinetic parameters of binding of the mutant to operator DNA reveals a 2000 to 3000-fold increase in the equilibrium dissociation constant (Kd) of the mutant-DNA complex in comparison to dimeric LacR-operator complexes, with the change almost entirely due to a greater than 1000-fold decrease in association rate. The dissociation rate varies only by a factor of about two, in comparison to dimeric LacR. This change reflects a kinetic pathway in which dimer formation, in solution or on DNA, is the rate-limiting step. These findings have implications for the specificity and stability of the protein-protein interface in question.

Four dimers of lambda repressor bound to two suitably spaced pairs of lambda operators form octamers and DNA loops over large distances.

Transcription factors that are bound specifically to DNA often interact with each other over thousands of base pairs [1] [2]. Large DNA loops resulting from such interactions have been observed in Escherichia coli with the transcription factors deoR [3] and NtrC [4], but such interactions are not, as yet, well understood. We propose that unique protein complexes, that are not present in solution, may form specifically on DNA. Their uniqueness would make it possible for them to interact tightly and specifically with each other. We used the repressor and operators of coliphage lambda to construct a model system in which to test our proposition. lambda repressor is a dimer at physiological concentrations, but forms tetramers and octamers at a hundredfold higher concentration. We predict that two lambda repressor dimers form a tetramer in vitro when bound to two lambda operators spaced 24 bp apart and that two such tetramers interact to form an octamer. We examined, in vitro, relaxed circular plasmid DNA in which such operator pairs were separated by 2,850 bp and 2,470 bp. Of these molecules, 29% formed loops as seen by electron microscopy (EM). The loop increased the tightness of binding of lambda repressor to lambda operator. Consequently, repression of the lambda PR promoter in vivo was increased fourfold by the presence of a second pair of lambda operators, separated by a distance of 3,600 bp.

The blood from Auschwitz and the silence of the scholars.

The Kaiser Wilhelm Institute for Anthropology, Human Genetics and Eugenics in Berlin-Dahlem was the centre of scientific racism in Nazi Germany. Its bad history culminated in a research project to analyse the molecular basis of racial differences in the susceptibility to various infectious diseases such as tuberculosis. Josef Mengele, a former postdoc of the director of the institute, Otmar von Verschuer, collected blood samples and other material in Auschwitz from families and twins of Jews and Gypsies. The blood samples were analysed by Günther Hillmann in the Berlin laboratory of Nobel Prize winner Adolf Butenandt. Butenandt had just moved to Tübingen. The project was paid for by the Deutsche Forschungsgemeinschaft. Butenandt, Hillmann and von Verschuer made scientific careers in the Federal Republic. To the present day this past has not been acknowledged by the Max-Planck-Gesellschaft as part of its history.

Dimeric lac repressors exhibit phase-dependent co-operativity.

Transcription of the lac operon in Escherichia coli is repressed by the binding of Lac repressor (LacR) to lac operator O1, a pseudo-palindromic sequence centred 11 bp downstream of the transcription start. Full repression of the wild-type promoter by wild-type, tetrameric LacR requires the presence of at least two operator sequences that must not only be in close proximity to O1, 401 bp and 92 bp for the auxiliary operators O2 and O3, respectively, but must also be present on the same side of the DNA helix. LacR mutants lacking the C-terminal heptad repeat and thus only capable of dimer formation still repress, but at a much reduced level. Their repression of the lac promoter is comparable to repression by tetrameric LacR when both auxiliary operators are destroyed. We have examined the residual repression, by dimeric LacR, of a series of constructs containing a CAP-independent promoter and two lac operators, O1 and Oid, separated by a series of spacers increasing in size by single base-pair increments. Surprisingly, repression of these constructs still exhibits phase dependence. The periodicity of maxima is similar to the helical repeat of DNA in vivo, as measured by phase-dependent repression with tetrameric LacR, although the magnitude of repression is much smaller than that obtained in previous experiments with tetrameric LacR. Two additional variants of dimeric LacR with altered C termini that were tested also show phase dependence. Control experiments show that the presence of O1 is required for repression in this system. In the absence of O1, occupancy of the auxiliary operator does not lead to repression. The magnitudes of repression maxima correlate best with the overall basic nature of the C terminus. Weak, unspecific contacts by this region with DNA seem sufficient to explain the observed periodicity. It remains to be seen whether additional factors are also involved in this residual repression.

Towards a mutant analysis of the tertiary structures of functional DNA-binding motifs.

Transcription factors have specific regions, often alpha helices, with which they recognise DNA. These regions are more or less disordered off DNA. Some examples are listed here. However, a detailed mutant analysis of this phenomenon is missing. It could show to what extent DNA binding in vitro and in vivo is harmed when such a region is artificially made rigid by suitable substitutions and could reveal how much transcription factors have improved by having been selected to carry unstable recognition domains.

The function of auxiliary operators.

Gene regulation by control of transcription has been analysed in great detail both in prokaryotes and in eukaryotes. The frequency of transcription may be decreased by repressors or increased by activators. A repressor may work by decreasing the concentration of RNA polymerase at a promoter capable of forming an open complex. An activator may work by increasing the concentration of RNA polymerase at a promoter capable of forming an open complex. For this purpose, a strategy is used over and over again. It is called increase in local concentration. How Escherichia coli uses this strategy efficiently is discussed.

Operator search by mutant Lac repressors.

The Escherichia coli Lac and Gal repressors are two members of a large family of bacterial repressor proteins that share significant sequence and structural homology. Efficient repression by all family members requires specific binding to a site or sites close to the transcriptional start of the genes regulated. Both LacR and GalR have to bind to at least two sites for efficient repression, yet they differ in one important respect: LacR is a homotetramer whereas GalR is a homodimer. In an attempt to understand this difference, we studied the operator binding activity of a LacR variant that has the DNA-binding specificity of GalR (LacR-V17A18). A tetrameric version of this protein shows a 30-fold decrease in association rate to operator located on a long (lambda) DNA molecule, in comparison to wild-type LacR, while a dimeric version of this protein shows an unaltered association rate in comparison to dimeric LacR. This reduction in association rate correlates with a broadened DNA-binding specificity for base-pairs 4 and 5 of the operator: examination of an additional LacR variant with an even broader DNA-binding specificity indicates that a tetrameric version also shows a 30-fold decrease in association rate in comparison to wild-type LacR, while a dimeric version again shows an unaltered association rate in comparison to dimeric LacR. This difference in association rate in vitro correlates with whether a tetrameric or dimeric variant of LacR of a given DNA-binding specificity will repress lacZ under control of a single operator more efficiently in vivo. We therefore propose that the formation of stable homotetramers becomes a distinct disadvantage unless a high degree of DNA-binding specificity is also present, and demonstrate that this in indeed the case for GalR-mediated repression of the gal operon. This functional constraint seems to have influenced the evolution of the LacI-GalR family of repressors, most of which have a relatively broad specificity of DNA-binding and most of which form only stable homodimers.

A positive selection vector for cloning of long polymerase chain reaction fragments based on a lethal mutant of the crp gene of Escherichia coli.

We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli. The positive selection pressure results from a lethal mutation within the E. coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this "suicide" vector, pCAPs, is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the lethal gene.

Enhanced GFAP expression in astrocytes of transgenic mice expressing the human brain-specific trypsinogen IV.

We recently identified a cDNA encoding a human brain specific trypsinogen (trypsinogen IV). In order to test whether trypsinogen IV is involved in CNS diseases of, or injury response in, mammalian brain, a mouse model was developed in which the human trypsinogen IV was expressed specifically in neurons. Immunocytochemical analysis of the brains of transgenic mice revealed a striking enhancement of glial fibrillar acidic protein (GFAP) expression in astrocytes. This remarkable astrocytic reaction was detected in the brains of mice as young as 2 months and did not diminish in the older animals we tested. However, we did not find gross evidence for neurodegeneration, nor for reactive microglial cells. The long-term survival of these animals should provide a model with which to study the mechanism of nerve-astroglia interactions. In addition, the possible participation of trypsin IV in the metabolism of the Alzheimer precursor protein (APP) was investigated by immunostaining brains from transgenic mice with beta-amyloid (betaA4) antibodies. Immunocytochemical staining of brains from one year old transgenic mice revealed an intense intracellular betaA4-like signal in neurons.

Some repressors of bacterial transcription.

For a long time, repression of transcription in Escherichia coli was thought to be generally caused by one repressor binding to one operator. Recent work has indicated the frequent presence of auxiliary operators and helper proteins. The recent solution of the X-ray structures of Lac and Pur repressors were breakthroughs; yet, it has become painfully clear that important aspects of repression are still not understood.