Quenching of bacteriochlorophyll a triplet state by carotenoids in the chlorosome baseplate of green bacterium Chloroflexus aurantiacus.

To capture weak light fluxes, green photosynthetic bacteria have unique structures - chlorosomes, consisting of 104-5 molecules of bacteriochlorophyll (BChl) c, d, e. Chlorosomes are attached to the cytoplasmic membrane through the baseplate, a paracrystalline protein structure containing BChl a and carotenoids (Car). The most important function of Car is the quenching of triplet states of BChl, which prevents the formation of singlet oxygen and thereby provides photoprotection. In our work, we studied the dynamics of the triplet states of BChl a and Car in the baseplate of Chloroflexus aurantiacus chlorosomes using picosecond differential spectroscopy. BChl a of the baseplate was excited into the Qy band at 810 nm, and the corresponding absorption changes were recorded in the range of 420-880 nm. It was found that the formation of the Car triplet state occurs in ∼1.3 ns, which is ∼3 times faster than the formation of this state in the peripheral antenna of C. aurantiacus according to literature data. The Car triplet state was recorded by the characteristic absorption band T1 → Tn at ∼550 nm. Simultaneously with the appearance of absorption T1 → Tn, there was a bleaching of the singlet absorption of Car in the region of 400-500 nm. Theoretical modeling made it possible to estimate the characteristic time of formation of the triplet state of BChl a as ∼0.5 ns. It is shown that the experimental data are well described by the sequential scheme of formation and quenching of the BChl a triplet state: BChl a* → BChl aT → CarT. Thus, carotenoids from green bacteria effectively protect the baseplate from possible damage by singlet oxygen.

Femtosecond Exciton Relaxation in Chlorosomes of the Photosynthetic Green Bacterium Chloroflexus aurantiacus.

Process of photosynthesis in the green bacteria Chloroflexus (Cfx.) aurantiacus starts from absorption of light by chlorosomes, peripheral antennas consisting of thousands of bacteriochlorophyll c (BChl c) molecules combined into oligomeric structures. In this case, the excited states are formed in BChl c, energy of which migrates along the chlorosome towards the baseplate and further to the reaction center, where the primary charge separation occurs. Energy migration is accompanied by non-radiative electronic transitions between the numerous exciton states, that is, exciton relaxation. In this work, we studied dynamics of the exciton relaxation in Cfx. aurantiacus chlorosomes using differential femtosecond spectroscopy at cryogenic temperature (80 K). Chlorosomes were excited by 20-fs light pulses at wavelengths in the range from 660 to 750 nm, and differential (light-dark) absorption kinetics were measured at a wavelength of 755 nm. Mathematical analysis of the obtained data revealed kinetic components with characteristic times of 140, 220, and 320 fs, which are responsible for exciton relaxation. As the excitation wavelength decreased, the number and relative contribution of these components increased. Theoretical modelling of the obtained data was carried out based of the cylindrical model of BChl c. Nonradiative transitions between the groups of exciton bands were described by a system of kinetic equations. The model that takes into account energy and structural disorder of chlorosomes turned out to be the most adequate.

Low-Frequency Oscillations of Bacteriochlorophyll Oligomers in Chlorosomes of Photosynthetic Green Bacteria.

In green photosynthetic bacteria, light is absorbed by bacteriochlorophyll (BChl) c/d/e oligomers, which are located in chlorosomes - unique structures created by Nature to collect the energy of very weak light fluxes. Using coherent femtosecond spectroscopy at cryogenic temperature, we detected and studied low-frequency vibrational motions of BChl c oligomers in chlorosomes of the green bacteria Chloroflexus (Cfx.) aurantiacus. The objects of the study were chlorosomes isolated from the bacterial cultures grown under different light intensity. It was found that the Fourier spectrum of low-frequency coherent oscillations in the Qy band of BChl c oligomers depends on the light intensity used for the growth of bacteria. It turned out that the number of low-frequency vibrational modes of chlorosomes increases as illumination under which they were cultivated decreases. Also, the frequency range within which these modes are observed expands, and frequencies of the most modes change. Theoretical modeling of the obtained data and analysis of the literature led to conclusion that the structural basis of Cfx. aurantiacus chlorosomes are short linear chains of BChl c combined into more complex structures. Increase in the length of these chains in chlorosomes grown under weaker light leads to the observed changes in the spectrum of vibrations of BChl c oligomers. This increase is an effective mechanism for bacteria adaptation to changing external conditions.

Dynamic Stark effect in ß and γ carotenes induced by photoexcitation of bacteriochlorophyll c in chlorosomes from Chloroflexus aurantiacus.

Chlorosomes of green bacteria can be considered as a prototype of future artificial light-harvesting devices due to their unique property of self-assembly of a large number of bacteriochlorophyll (BChl) c/d/e molecules into compact aggregates. The presence of carotenoids (Cars) in chlorosomes is very important for photoprotection, light harvesting and structure stabilization. In this work, we studied for the first time the electrochromic band shift (Stark effect) in Cars of the phototrophic filamentous green bacterium Chloroflexus (Cfx.) aurantiacus induced by fs light excitation of the main pigment, BChl c. The high accuracy of the spectral measurements permitted us to extract a small wavy spectral feature, which, obviously, can be associated with the dynamic shift of the Car absorption band. A global analysis of spectroscopy data and theoretical modeling of absorption spectra showed that near 60% of Cars exhibited a red Stark shift of ~ 25 cm-1 and the remaining 40% exhibited a blue shift. We interpreted this finding as evidence of various orientations of Car in chlorosomes. We estimated the average value of the light-induced electric field strength in the place of Car molecules as ~ 106 V/cm and the average distance between Car and the neighboring BChl c as ~ 10 Å. We concluded that the dynamics of the Car electrochromic band shift mainly reflected the dynamics of exciton migration through the chlorosome toward the baseplate within ~ 1 ps. Our work has unambiguously shown that Cars are sensitive indicators of light-induced internal electric fields in chlorosomes.

In memory of Vladimir Anatolievich Shuvalov (1943-2022): an outstanding biophysicist.

We present here a tribute to one of the foremost biophysicists of our time, Vladimir Anatolievich Shuvalov, who made important contributions in bioenergetics, especially on the primary steps of conversion of light energy into charge-separated states in both anoxygenic and oxygenic photosynthesis. For this, he and his research team exploited pico- and femtosecond transient absorption spectroscopy, photodichroism & circular dichroism spectroscopy, light-induced FTIR (Fourier-transform infrared) spectroscopy, and hole-burning spectroscopy. We remember him for his outstanding leadership and for being a wonderful mentor to many scientists in this area. Reminiscences by many [Suleyman Allakhverdiev (Russia); Robert Blankenship (USA); Richard Cogdell (UK); Arvi Freiberg (Estonia); Govindjee Govindjee (USA); Alexander Krasnovsky, jr, (Russia); William Parson (USA); Andrei Razjivin (Russia); Jian- Ren Shen (Japan); Sergei Shuvalov (Russia); Lyudmilla Vasilieva (Russia); and Andrei Yakovlev (Russia)] have included not only his wonderful personal character, but his outstanding scientific research.

Femtosecond excited-state dynamics in chlorosomal carotenoids of the photosynthetic bacterium Chloroflexus aurantiacus revealed by near infrared pump-probe spectroscopy.

In photosynthetic green bacteria, chlorosomes provide light harvesting with high efficiency. Chlorosomal carotenoids (Cars) participate in light harvesting together with the main pigment, bacteriochlorophyll (BChl) c/d/e. In the present work, we studied the excited-state dynamics in Cars from Chloroflexus (Cfx.) aurantiacus chlorosomes by near infrared pump-probe spectroscopy with 25 fs temporal resolution at room temperature. The S2 state of Cars was excited at a wavelength of ∼520 nm, and the absorption changes were probed at 860-1000 nm where the excited state absorption (ESA) of the Cars S2 state occurred. Global analysis of the spectroscopy data revealed an ultrafast (∼15 fs) and large (>130 nm) red shift of the S2 ESA spectrum together with the well-known S2 → S1 IC (∼190 fs) and Cars → BChl c EET (∼120 fs). The S2 lifetime was found to be ∼74 fs. Our findings are in line with earlier results on the excited-state dynamics in Cars in vitro. To explain the extremely fast S2 dynamics, we have tentatively proposed two alternative schemes. The first scheme assumed the formation of a vibrational wavepacket in the S2 state, the motion of which caused a dynamical red shift of the S2 ESA spectrum. The second scheme assumed the presence of two potential minima in the S2 state and incoherent energy transfer between them.

Utilization of blue-green light by chlorosomes from the photosynthetic bacterium Chloroflexus aurantiacus: Ultrafast excitation energy conversion and transfer.

Chlorosomes of photosynthetic green bacteria are unique molecular assemblies providing efficient light harvesting followed by multi-step transfer of excitation energy to reaction centers. In each chlorosome, 104-105 bacteriochlorophyll (BChl) c/d/e molecules are organized by self-assembly into high-ordered aggregates. We studied the early-time dynamics of the excitation energy flow and energy conversion in chlorosomes isolated from Chloroflexus (Cfx.) aurantiacus bacteria by pump-probe spectroscopy with 30-fs temporal resolution at room temperature. Both the S2 state of carotenoids (Cars) and the Soret states of BChl c were excited at ~490 nm, and absorption changes were probed at 400-900 nm. A global analysis of spectroscopy data revealed that the excitation energy transfer (EET) from Cars to BChl c aggregates occurred within ~100 fs, and the Soret â†’ Q energy conversion in BChl c occurred faster within ~40 fs. This conclusion was confirmed by a detailed comparison of the early exciton dynamics in chlorosomes with different content of Cars. These processes are accompanied by excitonic and vibrational relaxation within 100-270 fs. The well-known EET from BChl c to the baseplate BChl a proceeded on a ps time-scale. We showed that the S1 state of Cars does not participate in EET. We discussed the possible presence (or absence) of an intermediate state that might mediates the Soret â†’ Qy internal conversion in chlorosomal BChl c. We discussed a possible relationship between the observed exciton dynamics and the structural heterogeneity of chlorosomes.

Coherent intradimer dynamics in reaction centers of photosynthetic green bacterium Chloroflexus aurantiacus.

Early-time dynamics of absorbance changes (light minus dark) in the long-wavelength Qy absorption band of bacteriochlorophyll dimer P of isolated reaction centers (RCs) from thermophilic green bacterium Chloroflexus (Cfx.) aurantiacus was studied by difference pump-probe spectroscopy with 18-fs resolution at cryogenic temperature. It was found that the stimulated emission spectrum gradually moves to the red on the ~100-fs time scale and subsequently oscillates with a major frequency of ~140 cm-1. By applying the non-secular Redfield theory and linear susceptibility theory, the coherent dynamics of the stimulated emission from the excited state of the primary electron donor, bacteriochlorophyll dimer P*, was modeled. The model showed the possibility of an extremely fast transition from the locally excited state P1* to the spectrally different excited state P2*. This transition is clearly seen in the kinetics of the stimulated emission at 880 and 945 nm, where mostly P1* and P2* states emit, respectively. These findings are similar to those obtained previously in RCs of the purple bacterium Rhodobacter (Rba.) sphaeroides. The assumption about the existence of the second excited state P2* helps to explain the complicated temporal behavior of the ΔA spectrum measured by pump-probe spectroscopy. It is interesting that, in spite of the strong coupling between the P1* and P2* states assumed in our model, the form of the coherent oscillations is mainly defined by pure vibrational coherence in the excited states. A possible nature of the P2* state is discussed.

Q-band hyperchromism and B-band hypochromism of bacteriochlorophyll c as a tool for investigation of the oligomeric structure of chlorosomes of the green photosynthetic bacterium Chloroflexus aurantiacus.

Chlorosomes of green photosynthetic bacteria are the most amazing example of long-range ordered natural light-harvesting antennae. Chlorosomes are the largest among all known photosynthetic light-harvesting structures (~ 104-105 pigments in the aggregated state). The chlorosomal bacteriochlorophyll (BChl) c/d/e molecules are organized via self-assembly and do not require proteins to provide a scaffold for efficient light harvesting. Despite numerous investigations, a consensus regarding the spatial structure of chlorosomal antennae has not yet been reached. In the present work, we studied hyperchromism/hypochromism in the chlorosomal BChl c Q/B absorption bands of the green photosynthetic bacterium Chloroflexus (Cfx.) aurantiacus. The chlorosomes were isolated from cells grown under different light intensities and therefore, as we discovered earlier, they had different sizes of both BChl c antennae and their unit building blocks. We have shown experimentally that the Q-/B-band hyperchromism/hypochromism is proportional to the size of the chlorosomal antenna. We explained theoretically these findings in terms of excitonic intensity borrowing between the Q and B bands for the J-/H-aggregates of the BChls. The theory developed by Gülen (Photosynth Res 87:205-214, 2006) showed the dependence of the Q-/B-band hyperchromism/hypochromism on the structure of the aggregates. For the model of exciton-coupled BChl c linear chains within a unit building block, the theory predicted an increase in the hyperchromism/hypochromism with the increase in the number of molecules per chain and a decrease in it with the increase in the number of chains. It was previously shown that this model ensured a good fit with spectroscopy experiments and approximated the BChl c low packing density in vivo. The presented experimental and theoretical studies of the Q-/B-band hyperchromism/hypochromism permitted us to conclude that the unit building block of Cfx. aurantiacus chlorosomes comprises of several short BChl c chains.This conclusion is in accordance with previous linear and nonlinear spectroscopy studies on Cfx. aurantiacus chlorosomes.

Ultrafast excited-state dynamics in chlorosomes isolated from the photosynthetic filamentous green bacterium Chloroflexus aurantiacus.

Bacteriochlorophyll (BChl) c pigments in the aggregated state are responsible for efficient light harvesting in chlorosomes of the filamentous anoxygenic photosynthetic bacterium, Chloroflexus (Cfx.) aurantiacus. Absorption of light creates excited states in the BChl c aggregates. After subpicosecond intrachlorosomal energy transfer, redistribution and relaxation, the excitation is transferred to the BChl a complexes and further to reaction centers on the picosecond time scale. In this work, the femtosecond excited state dynamics within BChl c oligomers of isolated Cfx. aurantiacus chlorosomes was studied by double difference pump-probe spectroscopy at room temperature. Difference (Alight - Adark ) spectra corresponding to excitation at 725 nm (blue side of the BChl c absorption band) were compared with those corresponding to excitation at 750 nm (red side of the BChl c absorption band). A very fast (time constant 70 ± 10 fs) rise kinetic component was found in the stimulated emission (SE) upon excitation at 725 nm. This component was absent at 750-nm excitation. These data were explained by the dynamical red shift of the SE due to excited state relaxation. The nature and mechanisms of the ultrafast excited state dynamics in chlorosomal BChl c aggregates are discussed.

Variability of aggregation extent of light-harvesting pigments in peripheral antenna of Chloroflexus aurantiacus.

The stationary ground state and femtosecond time-resolved absorption spectra as well as spectra of circular dichroism were measured at room temperature using freshly prepared samples of chlorosomes isolated from fresh cultures of the green bacterium Chloroflexus aurantiacus. Cultures were grown by using as inoculum the same seed culture but under different light conditions. All measured spectra clearly showed the red shift of BChl c Qy bands (up to 5 nm) for low-light chlorosomes as compared to high-light ones, together with concomitant narrowing of these bands and increasing of their amplitudes. The sizes of the unit BChl c aggregates of the high-light-chlorosomes and the low-light ones were estimated. The fit of all experimental spectra was obtained within the framework of our model proposed before (Fetisova et al., Biophys J 71:995-101, 1996). The model assumes that a unit building block of the BChl c antenna has a form of a tubular aggregate of L = 6 linear single or double exciton-coupled pigment chains within a rod element, with the pigment packing density, approximating that in vivo. The simultaneous fit of all experimental spectra gave the number of pigments in each individual linear pigment chain N = 4 and N = 6 for the high-light and the low-light BChl c unit building blocks, respectively. The size of a unit building block in the BChl c antenna was found to vary from L × N = 24 to L × N = 36 exciton-coupled BChl c molecules being governed by the growth-light intensity. All sets of findings for Chloroflexus aurantiacus chlorosomes demonstrated the biologically expedient light-controlled variability, predicted by us, of the extent of BChl c aggregation within a unit building block in this antenna.

Orientation of B798 BChl a Q y transition dipoles in Chloroflexus aurantiacus chlorosomes: polarized transient absorption spectroscopy studies.

Isotropic and anisotropic pump-probe spectra of Cfx. aurantiacus chlorosomes were measured on the fs-through ps-time scales for the B798 BChl a Q y band upon direct excitation of the B798 band at T = 293 K and T = 90 K. Upon direct excitation of the B798 band, the anisotropy parameter value r(λ) was constant within the whole BChl a Q y band at any delay time at both temperatures. The value of the anisotropy parameter r decayed from r = 0.4 at both temperatures (at 200 fs delay time after excitation) to the steady-state values r = 0.1 at T = 293 K and to r = 0.09 at T = 90 K (at 30 ÷ 100 ps delay time after excitation). The results were considered within the framework of the model of uniaxial orientation distribution of BChl-a transition dipoles within a single Cfx. aurantiacus chlorosome. This implies that the B798 BChl a Q y transition dipoles, randomly distributed around the normal to the baseplate plane, form the angle θ with the plane. For this model, the theoretical dependence of the steady-state anisotropy parameter r on the angle θ was derived. According to the theoretical dependence r(θ), the angle θ corresponding to the experimental steady-state value r = 0.1 at T = 293 K was found to equal 55°. As the temperature drops to 90 K, the angle θ decreases to 54°.

Spectral exhibition of electron-vibrational relaxation in P* state of Rhodobacter sphaeroides reaction centers.

Electron-vibrational relaxation in the excited state of the primary electron donor, bacteriochlorophyll dimer P, in the reaction centers (RCs) of purple photosynthetic bacteria Rhodobacter sphaeroides is modeled. A multimode model of three states (i.e., the ground state Pg, initially excited P1*, and relaxed excited P2*) is used to calculate the incoherent dynamics of the difference (ΔA) spectra on a femtosecond timescale for the YM210 W mutant RCs. The relaxation processes are described by the step-ladder model. The model shows that the electron-vibrational relaxation in the excited state of P is visualized by the transient red shift of the stimulated emission from P*. The dynamics of this shift is observed as a change in the ΔA spectrum shape in its red-most part, within a few hundreds of femtoseconds after excitation. As a result, an initial rise in the red-side ΔA kinetics is delayed with respect to the blue-side kinetics. The time constant of the P1* â†’ P2* electronic relaxation (54 fs) and the Pg, P1*, and P2* vibrational relaxations (120 fs), used in the model, provided the best fit of the experimental time-resolved ΔA spectra and kinetics at 90 and 293 K. The possible nature of the P1* â†’ P2* electronic relaxation is discussed.

Limiting Distributions for Multitype Branching Processes.

In this paper the asymptotic behavior of multitype Markov branching processes with discrete or continuous time is investigated in the positive regular and nonsingular case when both the initial number of ancestors and the time tend to infinity. Some limiting distributions are obtained as well as multivariate asymptotic normality is proved. The paper considers also the relative frequencies of distinct types of individuals which is motivated by applications in the field of cell biology. We obtained non-random limits for the frequencies and multivariate asymptotic normality when the initial number of ancestors is large and the time of observation increases to infinity. In fact this paper continues the investigations of Yakovlev and Yanev [32] where the time was fixed. The new obtained limiting results are of special interest for cell kinetics studies where the relative frequencies but not the absolute cell counts are accessible to measurement.

Detecting intergene correlation changes in microarray analysis: a new approach to gene selection.

BACKGROUND: Microarray technology is commonly used as a simple screening tool with a focus on selecting genes that exhibit extremely large differential expressions between different phenotypes. It lacks the ability to select genes that change their relationships with other genes in different biological conditions (differentially correlated genes). We intend to enrich the above procedure by proposing a nonparametric selection procedure that selects differentially correlated genes. RESULTS: Using both simulations and resampling techniques, we found that our procedure correctly detected genes that were not differentially expressed but differentially correlated. We also applied our procedure to a set of biological data and found some potentially important genes that were not selected by the traditional method. DISCUSSION AND CONCLUSION: Microarray technology yields multidimensional information on the function of the whole genome. Rather than treating intergene correlation as a nuisance to the traditional gene selection procedures which are essentially univariate, our method utilizes the rich information contained in the correlation as a new selection criterion. It can provide additional useful candidate genes for the biologists.

Chromosome-specific spatial periodicities in gene expression revealed by spectral analysis.

Recent years have seen an unprecedented surge of research activity in studies of gene expression. This extensive work, however, has been almost uniformly focused on genome-wide gene expression and has largely ignored the fundamental fact that every gene has a specific chromosome location. We propose a novel method of spectral analysis for detecting hidden periodicities in gene expression signals ordered along the length of each chromosome. Using this method, we have discovered that each chromosome in rodents and humans has a unique periodic pattern of gene expression. The uncovered spatial periodicities in gene expression are tissue-specific in the sense that the largest differences in humans were observed between two normal tissues (brain and mammary gland) as well as between their tumor counterparts (glioma and breast cancer). The smallest differences resulted from the comparison of tumors (glioma and breast cancer) with their normal counterparts. All such effects do not extend to all chromosomes but are limited to only some of them. The estimated periods and amplitudes are identical for the genes located on the positive and negative DNA strands. While precise molecular mechanisms of chromosome-specific periodicities in gene expression have yet to be unraveled, their universal presence in different tissues adds another dimension to the current understanding of the genome organization.

Balancing Type One and Two Errors in Multiple Testing for Differential Expression of Genes.

A new procedure is proposed to balance type I and II errors in significance testing for differential expression of individual genes. Suppose that a collection, F(k), of k lists of selected genes is available, each of them approximating by their content the true set of differentially expressed genes. For example, such sets can be generated by a subsampling counterpart of the delete-d-jackknife method controlling the per-comparison error rate for each subsample. A final list of candidate genes, denoted by S(*), is composed in such a way that its contents be closest in some sense to all the sets thus generated. To measure "closeness" of gene lists, we introduce an asymmetric distance between sets with its asymmetry arising from a generally unequal assignment of the relative costs of type I and type II errors committed in the course of gene selection. The optimal set S(*) is defined as a minimizer of the average asymmetric distance from an arbitrary set S to all sets in the collection F(k). The minimization problem can be solved explicitly, leading to a frequency criterion for the inclusion of each gene in the final set. The proposed method is tested by resampling from real microarray gene expression data with artificially introduced shifts in expression levels of pre-defined genes, thereby mimicking their differential expression.

Wave packet motions coupled to electron transfer in reaction centers of Chloroflexus aurantiacus.

Transient absorption difference spectroscopy with approximately 20 femtosecond (fs) resolution was applied to study the time and spectral evolution of low-temperature (90 K) absorbance changes in isolated reaction centers (RCs) of Chloroflexus (C.) aurantiacus. In RCs, the composition of the B-branch chromophores is different with respect to that of purple bacterial RCs by occupying the B(B) binding site of accessory bacteriochlorophyll by bacteriopheophytin molecule (Phi(B)). It was found that the nuclear wave packet motion induced on the potential energy surface of the excited state of the primary electron donor P* by approximately 20 fs excitation leads to a coherent formation of the states P+Phi(B)(-) and P+B(A)(-) (B(A) is a bacteriochlorophyll monomer in the A-branch of cofactors). The processes were studied by measuring coherent oscillations in kinetics of the absorbance changes at 900 nm and 940 nm (P* stimulated emission), at 750 nm and 785 nm (Phi(B) absorption bands), and at 1,020-1028 nm (B(A)(-) absorption band). In RCs, the immediate bleaching of the P band at 880 nm and the appearance of the stimulated wave packet emission at 900 nm were accompanied (with a small delay of 10-20 fs) by electron transfer from P* to the B-branch with bleaching of the Phi(B) absorption band at 785 nm due to Phi(B)(-) formation. These data are consistent with recent measurements for the mutant HM182L Rb. sphaeroides RCs (Yakovlev et al., Biochim Biophys Acta 1757:369-379, 2006). Only at a delay of 120 fs was the electron transfer from P* to the A-branch observed with a development of the B(A)(-) absorption band at 1028 nm. This development was in phase with the appearance of the P* stimulated emission at 940 nm. The data on the A-branch electron transfer in C. aurantiacus RCs are consistent with those observed in native RCs of Rb. sphaeroides. The mechanism of charge separation in RCs with the modified B-branch pigment composition is discussed in terms of coupling between the nuclear wave packet motion and electron transfer from P* to Phi(B) and B(A) primary acceptors in the B-branch and A-branch, respectively.

A nitty-gritty aspect of correlation and network inference from gene expression data.

BACKGROUND: All currently available methods of network/association inference from microarray gene expression measurements implicitly assume that such measurements represent the actual expression levels of different genes within each cell included in the biological sample under study. Contrary to this common belief, modern microarray technology produces signals aggregated over a random number of individual cells, a "nitty-gritty" aspect of such arrays, thereby causing a random effect that distorts the correlation structure of intra-cellular gene expression levels. RESULTS: This paper provides a theoretical consideration of the random effect of signal aggregation and its implications for correlation analysis and network inference. An attempt is made to quantitatively assess the magnitude of this effect from real data. Some preliminary ideas are offered to mitigate the consequences of random signal aggregation in the analysis of gene expression data. CONCLUSION: Resulting from the summation of expression intensities over a random number of individual cells, the observed signals may not adequately reflect the true dependence structure of intra-cellular gene expression levels needed as a source of information for network reconstruction. Whether the reported effect is extrime or not, the important point, is to reconize and incorporate such signal source for proper inference. The usefulness of inference on genetic regulatory structures from microarray data depends critically on the ability of investigators to overcome this obstacle in a scientifically sound way. REVIEWERS: This article was reviewed by Byung Soo KIM, Jeanne Kowalski and Geoff McLachlan.

Synergistic response to oncogenic mutations defines gene class critical to cancer phenotype.

Understanding the molecular underpinnings of cancer is of critical importance to the development of targeted intervention strategies. Identification of such targets, however, is notoriously difficult and unpredictable. Malignant cell transformation requires the cooperation of a few oncogenic mutations that cause substantial reorganization of many cell features and induce complex changes in gene expression patterns. Genes critical to this multifaceted cellular phenotype have therefore only been identified after signalling pathway analysis or on an ad hoc basis. Our observations that cell transformation by cooperating oncogenic lesions depends on synergistic modulation of downstream signalling circuitry suggest that malignant transformation is a highly cooperative process, involving synergy at multiple levels of regulation, including gene expression. Here we show that a large proportion of genes controlled synergistically by loss-of-function p53 and Ras activation are critical to the malignant state of murine and human colon cells. Notably, 14 out of 24 'cooperation response genes' were found to contribute to tumour formation in gene perturbation experiments. In contrast, only 1 in 14 perturbations of the genes responding in a non-synergistic manner had a similar effect. Synergistic control of gene expression by oncogenic mutations thus emerges as an underlying key to malignancy, and provides an attractive rationale for identifying intervention targets in gene networks downstream of oncogenic gain- and loss-of-function mutations.