Mammalian genes as molecular clocks?

Summary In analyzing the silent nucleotide substitutions in some mammalian mitochondrial mRNA coding genes, we had found that the frequency of each of the four nucleotides in rat, mouse, and cow, but not in humans, is the same in the silent third codon position (Lanave C, Preparata G, Saccone C, Serio G (1984) J Mol Evol 20:86-93). Because our findings for these three species were compatible with a stationary Markov process for the evolution of nucleotide sequences, we applied such a model to calculate the effective evolutionary silent substitution rate (v_s) and the divergence times among the species. In this paper we have analyzed the first and second codon positions in the same mammalian mitochondrial genes. We found that in the first and second codon positions the human mitochondrial genes satisfy the stationarity conditions. This has allowed us to use the stochastic model mentioned above to calculate the divergence times among mouse, rat, cow, and human. Furthermore, we have analyzed the silent substitution rate in one nuclear gene for these four mammals. We found that in this gene the effective silent substitution rate is about 3 times lower than in mitochondrial genes, and that humans are in this case stationary with respect to the other three mammals in the third codon position as well. Application of our Markov model to this latter gene yields divergence times consistent with our previous determinations.     

How to identify essential genes from molecular networks?

Background  

The prediction of essential genes from molecular networks is a way to test the understanding of essentiality in the context of what is known about the network. However, the current knowledge on molecular network structures is incomplete yet, and consequently the strategies aimed to predict essential genes are prone to uncertain predictions. We propose that simultaneously evaluating different network structures and different algorithms representing gene essentiality (centrality measures) may identify essential genes in networks in a reliable fashion.     

Do nucleic acids moonlight as molecular chaperones?

Organisms use molecular chaperones to combat the unfolding and aggregation of proteins. While protein chaperones have been widely studied, here we demonstrate that DNA and RNA exhibit potent chaperone activity in vitro. Nucleic acids suppress the aggregation of classic chaperone substrates up to 300-fold more effectively than the protein chaperone GroEL. Additionally, RNA cooperates with the DnaK chaperone system to refold purified luciferase. Our findings reveal a possible new role for nucleic acids within the cell: that nucleic acids directly participate in maintaining proteostasis by preventing protein aggregation.     

Are all genes regulatory genes?

Although much has been learned about hereditary mechanisms since Gregor Mendel’s famous experiments, gene concepts have always remained vague, notwithstanding their central role in biology. During over hundred years of genetic research, gene concepts have often and dynamically changed to accommodate novel experimental findings, without ever providing a generally accepted definition of the ‘gene.’ Yet, the distinction between ‘regulatory genes’ and ‘structural genes’ has remained a common theme in modern gene concepts since the definition of the operon-model. This distinction is now challenged by recent findings which suggest that, at least in eukaryotes, structural genes may in many situations have a regulatory function that is independent of the function of the gene product (protein or non-coding RNA molecule). This brief paper discusses these new findings and some possible implications for the notion of the ‘regulatory gene.’     

Pollen as a vehicle for the escape of engineered genes?

Recent studies of pollen exchange between neighboring populations of plants have shown that interpopulation gene flow can proceed over much greater distances and at higher rates than hitherto believed. This means that the escape of engineered genes from crop plants to their wild relatives is not only possible, but also likely. The development of containment strategies, such as extra modifications for increased self-fertilization and decreased pollen longevity in engineered crop plants, will be necessary to safeguard against such escape.     

Does genetic conflict drive rapid molecular evolution of nuclear transport genes in Drosophila?

The Segregation Distorter (SD) system of Drosophila melanogaster is one the best-characterized meiotic drive complexes known. SD gains an unfair transmission advantage through heterozygous SD/SD(+) males by incapacitating SD(+)-bearing spermatids so that virtually all progeny inherit SD. Segregation distorter (Sd), the primary distorting locus in the SD complex, is a truncated duplication of the RanGAP gene, a major regulator of the small GTPase Ran, which has several functions including the maintenance of the nucleocytoplasmic RanGTP concentration gradient that mediates nuclear transport. The truncated Sd-RanGAP protein is enzymatically active but mislocalizes to the nucleus where it somehow causes distortion. Here I present data consistent with the idea that wild-type RanGAP, and possibly other loci able to influence the RanGTP gradient, has been caught up in an ancient genetic conflict that predates the SD complex. The legacy of this conflict could include the unexpectedly rapid evolution of nuclear transport-related proteins, the accumulation of chromosomal inversions, the recruitment of gene duplications, and the turnover of repetitive sequences in the centric heterochromatin.     

Circadian clocks: what makes them tick?

In the not too distant past, it was common belief that rhythms in the physical environment were the driving force, to which organisms responded passively, for the observed daily rhythms in measurable physiological and behavioral variables. The demonstration that this was not the case, but that both plants and animals possess accurate endogenous time-measuring machinery (i.e., circadian clocks) contributed to heightening interest in the study of circadian biological rhythms. In the last few decades, flourishing studies have demonstrated that most organisms have at least one internal circadian timekeeping device that oscillates with a period close to that of the astronomical day (i.e., 24h). To date, many of the physiological mechanisms underlying the control of circadian rhythmicity have been described, while the improvement of molecular biology techniques has permitted extraordinary advancements in our knowledge of the molecular components involved in the machinery underlying the functioning of circadian clocks in many different organisms, man included. In this review, we attempt to summarize our current understanding of the genetic and molecular biology of circadian clocks in cyanobacteria, fungi, insects, and mammals. (Chronobiology International, 17(4), 433-451, 2000)     

Are there multiple circadian clocks in plants?

We have reported that Arabidopsis might have genetically distinct circadian oscillators in multiple cell-types.¹ Rhythms of CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter activity are 2.5 h longer in phytochromeB mutants in constant red light and in cryptocrome1 cry2 double mutant (hy4-1 fha-1) in constant blue light than the wild-type.² However, we found that cytosolic free Ca²⁺ ([Ca²⁺]_cyt) oscillations were undetectable in these mutants in the same light conditions.¹ Furthermore, mutants of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) have short period rhythms of leaf movement but have arrhythmic [Ca²⁺]_cyt oscillations. More important, the timing of cab1-1 (toc1-1) mutant has short period rhythms of CAB2 promoter activity (∼21 h) but, surprisingly, has a wild-type period for circadian [Ca²⁺]_cyt oscillations (∼24 h). In contrast, toc1-2, a TOC1 loss-of-function mutant, has a short period of both CAB2 and [Ca²⁺]_cyt rhythms (∼21 h). Here we discuss the difference between the phenotypes of toc1-1 and toc1-2 and how rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations might be regulated differently.Key words: circadian rhythms, TOC1, multiple oscillators, CAB2, Ca²⁺ signalling, arabidopsis, circadian [Ca²⁺]_cyt oscillations, aequorin, luciferase, central oscillatorThe plant circadian clock controls a multitude of physiological processes such as photosynthesis, organ and stomatal movements and transition to reproductive growth. A plant clock that is correctly matched to the rhythms in the environment brings about a photosynthetic advantage that results in more chlorophyll, more carbon assimilation and faster growth.³ One of the first circadian clock mutants to be described in plants was the short period timing of cab1-1 (toc1-1), which was identified using the rhythms of luciferase under a CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter as a marker for circadian period.⁴Circadian rhythms of both CAB2 promoter activity and cytosolic-free Ca²⁺ ([Ca²⁺]_cyt) oscillations depend on the function of a TOC1, CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL (TOC1/CCA1/LHY) negative feedback loop.⁵ In tobacco seedlings, CAB2:luciferase (CAB2:luc) rhythms and circadian [Ca²⁺]_cyt oscillations can be uncoupled in undifferentiated calli.⁶ In Arabidopsis, we reported that toc1-1 has different periods of rhythms of CAB2 promoter activity (∼21 h) and circadian [Ca²⁺]_cyt oscillations (∼24 h). The mutant allele toc1-1 has a base pair change that leads to a full protein that has an amino acid change from Ala to Val in the CCT domain (CONSTANS, CONSTANS-LIKE and TOC1).⁷ On the other hand, the mutant toc1-2 has short period of both rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations (∼21 h).^1,7 This allele has a base pair change that results in changes to preferential mRNA splicing, resulting in a truncated protein with only 59 residues.⁷ Thus, the mutated CCT domain in toc1-1 might lead to the uncoupling of rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations while the absence of TOC1 in toc1-2 causes the shortening of the period of both rhythms. Indeed, zeitlupe-1 (ztl-1) mutants, that have higher levels of TOC1, have long periods of both rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations.¹ The biochemical function of the CCT domain is unknown but it is predicted to play an important role in protein-protein interactions⁸ and nuclear localization.⁹One model to explain the period difference of CAB2:luc expression and circadian [Ca²⁺]_cyt oscillation is that the toc1-1 mutation has uncoupled two oscillators in the same cell. Uncoupled oscillators are a predicted outcome of certain mutations in the recently described three-loop mathematical model.^10–11 However, both rhythms of TOC1 and CCA1/LHY expression, which would be in uncoupled oscillators accordingly to the model, are described as short-period in toc1-1.⁵ Thus, we have favored the model in which CAB2:luc expression and circadian [Ca²⁺]_cyt oscillation are reporting cell-types with different oscillators that are affected differently by toc1-1.It is possible that TOC1 could interact with a family of cell-type specific proteins. The interaction of TOC1 with each member of the family could be affected differently by the mutation in the CCT domain (Fig. 1). Two-hybrid assays have shown that TOC1 interacts with PIF proteins (PHYTOCHROME INTERACTING FACTOR3 and PIF4) and related PIL proteins (PIF3-LIKE PROTEIN 1, PIL2, PIL5 and PIL6).⁸ In fact, TOC1 interaction with both PIF3 and PIL1 is stronger when the N-terminus receiver domain is taken out and the CCT domain is left intact.⁸ Thus, it is possible that TOC1 and different PIF/PIL proteins interact to regulate the central oscillator. This interaction could be impaired by the Ala to Val change in the toc1-1 mutation, leading to the period shortening. However, lines misexpressing PIF3, PIL1 and PIL6 showed no changes in their circadian rhythms.^12–16Open in a separate windowFigure 1Models of how the toc1-1 mutation might differently affect cell-type specific circadian oscillators. The single mutant toc1-1 have 21 h rhythms of CAB2 promoter activity and 24 h-rhythms of [Ca²⁺]_cyt oscillations. The toc1-1 mutation is a single amino acid change in the CCT domain. The CCT domain is involved in protein-protein interaction and/or nuclear localization. We have proposed that circadian oscillators with different periods are present in different cell-types. The luminescence generated by CAB2 promoter-drived luciferase (from the CAB2:luc) is probably originated in the epidermis and mesophyll cells. In this model, we propose that the mutation on the CCT domain impairs the mutated TOC1 interaction with the hypothetical protein Z in these cells-types. In contrast, in other cell-types, the mutated TOC1 still interacts with other hypothetical proteins (W), despite the mutation in the CCT domain. In those cell-types, the circadian oscillator could still run with a 24 h period for [Ca²⁺]_cyt rhythms (from the 35S:AEQ construct). One possible identity for Z and W are the members of the PHYTOCHROME INTERACTING FACTOR (PIF) related PIF3-LIKE (PIL) family.One possible explanation for the absence of alterations in the period of circadian rhythms in lines misexpressing PIF/PIL is that they only have roles in certain cell-types. As an example, PIL6 and PIF3 are involved with flowering time and hypocotyl growth in red light^12–15 while PIL1 and PIL2 are involved with hypocotyl elongation in shade-avoidance responses.¹⁶ Both hypocotyl growth and flowering time require cell-type specific regulation: vascular bundle cells in the case of the flowering time¹⁷ and the cells in the shoot in the case of the hypocotyl elongation.¹⁶ If TOC1 interaction with certain PIF/PIL is indeed cell-type specific, the mutated CCT domain found in the toc1-1 mutant could affect the clock in different ways, depending on the type of PIF/PIL protein expressed in each cell-type. Therefore, a question that arises is: which cell-types are sensitive to the toc1-1 mutation?There is evidence that CAB2 and CATALASE3 (CAT3) are regulated by two oscillators that respond differently to temperature signals.¹⁸ These genes might be regulated by two distinct circadian oscillators within the same tissues or a single cell.¹⁸ Interestingly, the spatial patterns of expression of CAB2 and CATALASE3 overlap in the mesophyll of the cotyledons.¹⁸ Furthermore, rhythms of CAB2 and CHALCONE SYNTHASE (CHS) promoter activity have different periods and they are equally affected by toc1-1 mutation.¹⁹ Whereas CAB2 is mainly expressed in the mesophyll cells, CHS is mainly expressed in epidermis and root cells.¹⁹ However, rhythms of AEQUORIN luminescence, which reports [Ca²⁺]_cyt oscillation, were insensitive to toc1-1 mutation and appear to come from the whole cotyledon.²⁰ One cell-type which is found in the whole cotyledon but is distinct from either mesophyll or epidermis cells is the vascular tissue and associated cells.Another approach to determine which cell-types are insensitive to toc1-1 mutation is to compare the toc1-1 and toc1-2 phenotypes. The period of circadian [Ca²⁺]_cyt oscillations is not the only phenotype that is different in toc1-1 and toc1-2 mutants. Rhythms in CAB2 promoter activity in constant red light are short period in toc1-1 but arrhythmic in toc1-2.^21,22 COLD, CIRCADIAN RHYTHM AND RNA BINDING 2/GLYCINE-RICH RNA BINDING PROTEIN 7 (CCR2/GRP7) is also arrhythmic in toc1-2 but short period in toc1-1 in constant darkness.^7,22 When the length of the hypocotyl was measured for both toc1-1 and toc1-2 plants exposed to various intensities of red light, only toc1-2 had a clear reduction in sensitivity to red light. Therefore, toc1-2 has long hypocotyl when maintained in constant red light while hypocotyl length in toc1-1 is nearly identical to that in the wild-type.²² These differences may allow us to separate which cell-types are sensitive to the toc1-1 mutation and which not.Hypocotyl growth is regulated by a large number of factors such as light, gravity, auxin, cytokinins, ethylene, gibberellins and brassinosteroids.²³ There is also a correlation between the size of the hypocotyl in red light and defects in the circadian signaling network.^24,25 The fact that toc1-1 has different hypocotyl sizes from toc1-2 suggests that circadian [Ca²⁺]_cyt oscillations could be involved in the light-dependent control of hypocotyl growth. Circadian [Ca²⁺]_cyt oscillations might encode temporal information to control cell expansion and hypocotyl growth.^26–28 toc1-1 have short-period rhythms of hypocotyl elongation, which indicates that the cells in the hypocotyl have a 21 h oscillator.²⁹ However, toc1-1 might also have a wild-type hypocotyl length in continuous red light because cells which generate the signal to regulate hypocotyl growth might have 24 h oscillators.The toc1-1 mutation was the first to be directly associated with the plant circadian clock, revitalizing the field of study.⁴ Now, by either uncoupling two feedback loops or by distinct TOC1 protein-protein interaction in different cell-types, toc1-1 has shown new properties of the circadian clock that may deepen our understanding of this system.     

Circadian clocks: A cry in the dark?

Cryptochrome proteins are key components of the circadian systems of both Drosophila and mammals. In Drosophila, they appear to be responsible for the entrainment of the circadian clock by the light-dark cycle, while in mammals they perform an important role in rhythm generation itself.     

How similar are daily and seasonal biological clocks?

Daily and seasonal timing systems in insects have usually been supposed to share similar mechanisms, because both rely in large measure on information from the daily light-dark cycle: daily clocks can ensure that activity coincides with the appropriate time of day, and seasonal time is indicated most reliably by daylength. However, several lines of evidence suggest that the systems are different. For example, receptor features, photosensitive pigments, clocks, and the effectors that mediate responses to information derived from the clock may have different daily, seasonal and general functions and properties, and several different systems are known. There are many different additional elements in the seasonal response. Therefore, these responses may not rely on similar timing mechanisms, despite the long-standing belief that the seasonal clock has circadian components. Such a difference would be consistent with the fact that temporal responses serve a very wide range of purposes, meeting many different ecological needs on different time frames. Consequently, understanding the seasonal relevance of the photoperiodic responses is more important than revealing any possible involvement with circadian systems.     

More genes in vertebrates?

With the acquisition of complete genome sequences from several animals, there is renewed interest in the pattern of genome evolution on our own lineage. One key question is whether gene number increased during chordate or vertebrate evolution. It is argued here that comparing the total number of genes between a fly, a nematode and human is not appropriate to address this question. Extensive gene loss after duplication is one complication; another is the problem of comparing taxa that are phylogenetically very distant. Amphioxus and tunicates are more appropriate animals for comparison to vertebrates. Comparisons of clustered homeobox genes, where gene loss can be identified, reveals a one to four mode of evolution for Hox and ParaHox genes. Analyses of other gene families in amphioxus and vertebrates confirm that gene duplication was very widespread on the vertebrate lineage. These data confirm that vertebrates have more genes than their closest invertebrate relatives, acquired through gene duplication. abbreviations IHGSC, International Human Genome Sequencing Consortium; TCESC, The C. elegans Sequencing Consortium.     

What is a pheromone? Mammalian pheromones reconsidered

Pheromone communication is a two-component system: signaling pheromones and receiving sensory neurons. Currently, pheromones remain enigmatic bioactive compounds, as only a few have been identified, but classical bioassays have suggested that they are nonvolatile, activate vomeronasal sensory neurons, and regulate innate social behaviors and neuroendocrine release. Recent discoveries of potential pheromones reveal that they may be more structurally and functionally diverse than previously defined.