Science Research Ideas
Genetics
2010:
“Different mechanisms and models have been proposed for such divergence(how one gene diverges to give two paralogous genes) processes, and these mechanisms differ by assuming that different timings and different selection forces act on the starting gene versus its duplicated copy. However, the relevance and feasibility of these various models is still unclear.” (Soskine & Tawfik, 2010)
“Divergence of sequence and function also occurs without duplication (of genes). Sequence divergence occurs constantly and gives rise to orthologues, the functions of which are considered to be identical. Many of the sequence differences between orthologues are therefore considered neutral — that is, the sequence differences are not associated with adaptation towards new functions. The degree to which these assumptions are true is unclear.” (Soskine & Tawfik, 2010)
“Although our knowledge of how mutations affect protein structure and function, and subsequently protein evolution, has increased dramatically, many aspects remain unexplored.” (Soskine & Tawfik, 2010)
“The distribution of mutational effects — beyond single proteins: The fitness effects of mutations in a cellular, let alone organismal, context are much more complex than in isolated proteins such as TEM1 β-lactamase. Future aims include the examination of fitness effects under native conditions (such as the expression of a chromosomal gene from its endogenous promoter) and the investigation of how the fate of mutated variants is affected by various buffering mechanisms and by mechanisms of protein trafficking and clearance.” (Soskine & Tawfik, 2010)
“The distribution of mutational effects — is an organism a sum of its proteins? Surprisingly, on preliminary examination, organisms seem to be more sensitive to mutations than their component proteins. The overall similarity in the distributions of fitness effects of mutations for a single protein and an intact organism, in addition to the differences, demand further exploration.” (Soskine & Tawfik, 2010)
“Studies of natural protein divergence: Our knowledge of protein mutations and their effects comes primarily from the study of model cases. In only a few cases has the natural divergence of a protein (or its clinical divergence, as in TEM1) been subsequently studied in the laboratory. Studies of actual adaptations will provide an interesting and more conclusive picture of how new genes and proteins diverge. Potential study targets include adaptations towards man-made chemicals, such as pesticides and herbicides in bacteria, insectsor plants. Secondary metabolism in plants is another rich source of functional diversification.” (Soskine & Tawfik, 2010)
“Reconstructions of ancestors and their divergence paths: Reconstructing ancestral genes and proteins and the divergence paths that led to contemporary proteins is a powerful approach that can also be applied to examine the mutational paths and the effects of individual mutations on the divergence process.” (Soskine & Tawfik, 2010)
“Compartmental adaptations: ‘Function’ refers not only to what a protein does in a living organism but also to where it does it. The same activity (for example, catalysing a given enzymatic transformation) can take place in different compartments or organisms. This means that proteins can be placed under different regulation schemes and be processed differently with regard to synthesis, transport, pH optimum, stability, and so on. It will be interesting to explore the sequence changes that drive compartmental adaptation, as these could be more intense than the sequence alterations that drive changes in activity itself.” (Soskine & Tawfik, 2010)
“Divergence by horizontal gene transfer: Horizontal (lateral) gene transfer is a common source of evolutionary innovation. A related gene imported from another organism partly resembles a newly formed duplicate, so it will be worth investigating whether the above-discussed mechanisms, mutational effects and their trade-offs apply.” (Soskine & Tawfik, 2010)
“Evolutionary rates: The rates of evolution of proteins (the average number of amino acid exchanges per position, per generation) are widely distributed between organisms (viral proteins provide a clear example), as well as within the same organism. Many factors might be involved, but the effects of the protein’s structure and its response to mutations (the distribution of mutational effects) remain unclear.” (Soskine & Tawfik, 2010)
“Epistasis and protein evolution: Although it is beyond the scope of this Review, epistasis is an important factor in protein evolution. If the effect of a given mutation depends on whether another mutation (or mutations) is present, the likelihood and mechanism of divergence will be affected. For example, new-function mutations may not be fixed unless a stabilizing, compensatory mutation is already present. Conversely, a compensatory mutation can be neutral or even deleterious on its own but beneficial in combination with a destabilizing mutation. Exploring the mutations that underline various divergence paths may provide new insights regarding the role of epistasis in directing the mechanism of divergence.” (Soskine & Tawfik, 2010)
“Other RdDM components, including DRM2, ARGONAUTE 4 (AGO4) and Pol V, are needed for siRNA accumulation for a subset of loci; however, these proteins do not seem to be involved in the initial production of siRNAs and are proposed to reinforce siRNA biogenesis by an unknown mechanism.” (Law & Jacobsen, 2010)
“The precise relationship among Pol II, Pol V and Pol IV remains elusive, but these studies suggest they may be more intimately connected than previously thought.” (Law & Jacobsen, 2010)
“ DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1), a putative chromatin-remodelling factor, and DMS3, an RdDM component with similarity to structural maintenance of chromosome (SMC) proteins, are needed for NRPE1 (NUCLEAR RNA POLYMERASE E1)chromatin association and for accumulation of IGN transcripts, although how these components are targeted is unknown.” (Law & Jacobsen, 2010)
“As DNMT3A seems to be a non-processive DNA methyltransferase, the formation of an oligomer could help to explain the observed periodic pattern of DNA methylation. Whether oligomerization occurs in vivo remains unknown, but it is tempting to propose a model in which interactions between DNMT3L and unmethylated H3K4 tails, or possibly between DNMT3A and other histone modifications or histone methyltransferases, might target and set the register for oligomerization of tetramers consisting of DNMT3A and DNMT3L, resulting in an ~8–10 bp periodicity.” (Law & Jacobsen, 2010)
“It was postulated that siRNAs generated in the vegetative nucleus might travel to the sperm cells and reinforce silencing by an unknown mechanism.” (Law & Jacobsen, 2010)
“Early studies of mammalian piRNA populations revealed that, unlike in D. melanogaster, mammalian piRNAs were not enriched for repetitive regions of the genome, leaving it unclear whether mammalian piRNAs function to silence transposons.” (Law & Jacobsen, 2010)
“Increases in antisense transcripts in met1 mutants were found to be rare and uncorrelated with body methylated genes9. Therefore, the function of body methylation remains poorly understood.” (Law & Jacobsen, 2010)
“The mechanism through which LSH1 functions in DNA methylation remains unknown.” (Law & Jacobsen, 2010)
“Whether direct protein interactions between CMT3 (Chromomethylase-3)and SUVH4 (SUPPRESSOR OF VARIEGATION 3-9 HOMOLOGUE 4)occur and aid in maintaining CHG methylation in plants is unknown.” (Law & Jacobsen, 2010)
“In mammals, the resulting single-nucleotide gap is then acted on by DNA polymerase-β and DNA ligase IIIα to repair the DNA through the short-patch BER pathway. Homologues of these enzymes have not been identified in plants, raising the possibility that plants use enzymes that are involved in the long-patch BER (base excision repair) pathway.” (Law & Jacobsen, 2010)
“After reciprocal crosses between two A. thaliana ecotypes (A genetically distinct population within a widely spread species.), siRNAs from the resultant silique (An elongated seed capsule that is formed after fertilization.) tissue (whichcontains the developing embryos) were sequenced, andnearly all of the Pol IV-dependent siRNAs that could be uniquely distinguished between the two ecotypeswere maternal in origin. What causes these loci to be maternally imprinted, whether this imprinting requiresDME and what function this massive extent of imprintingserves remain unknown. One hypothesis is that suchmaternal imprinting would allow recognition of selffrom non-self and have a suppressive effect on hybrids (Offspring that are produced by crossing two different populations within a single species).” (Law & Jacobsen, 2010)
“The mechanism (or mechanisms) through which the DME/ROS1 glycosylases are targeted to specific loci to carry out DNA demethylation is (are) unknown. These A. thaliana glycosylases are quite different from most other glycosylases: they are much larger and contain two conserved domains of unknown function. Whether these domains are required for targeting demethylation remains unknown.” (Law & Jacobsen, 2010)
“Proteins that are orthologous to the DME/ROS1 family of glycosylases have not been identified in mammals, and the existence of other enzymes that can directly remove methylated cytosines is controversial.” (Law & Jacobsen, 2010)
“In vivo, the observed decrease in methylation at the CYP27B1(cytochrome p450 27B1) promoter can occur in the absence of DNA replication — which suggests an active mechanism — and is dependent on the presence of a catalytically active MBD4 protein (an HhH-GPD thymine glycosylase that is related to the A. thaliana DME/ROS1 family of glycosylases and that has active mammalian homologues) that contains the serine residues that are targeted for phosphorylation. These recent findings need to be confirmed, and whether such a mechanism for the direct removal of methylated cytosines could account for DNA demethylation on a larger scale remains unknown.” (Law & Jacobsen, 2010)
“Despite the significant advances in our understanding of DNA methylation pathways, several key questions remain, especially surrounding the issue of targeting. How DNA methyltransferases are targeted by siRNAs and piRNAs in plants and mammals, respectively, remains elusive. In terms of DNA demethylation, whether DME is specifically targeted to many sites throughout the genome during gametogenesis or whether it non-selectively removes methylation remains unclear. Similarly, whether demethylation by the other DME/ROS1 family members is specifically directed to certain loci or whether the observed methylation pattern simply reflects a balance between the RdDM and demethylation pathways requires further investigation. Gaining further insights into mammalian DNA demethylation pathways and understanding how demethylation is targeted will be key challenges for future research.” (Law & Jacobsen, 2010)
“Different mechanisms and models have been proposed for such divergence(how one gene diverges to give two paralogous genes) processes, and these mechanisms differ by assuming that different timings and different selection forces act on the starting gene versus its duplicated copy. However, the relevance and feasibility of these various models is still unclear.” (Soskine & Tawfik, 2010)
“Divergence of sequence and function also occurs without duplication (of genes). Sequence divergence occurs constantly and gives rise to orthologues, the functions of which are considered to be identical. Many of the sequence differences between orthologues are therefore considered neutral — that is, the sequence differences are not associated with adaptation towards new functions. The degree to which these assumptions are true is unclear.” (Soskine & Tawfik, 2010)
“Although our knowledge of how mutations affect protein structure and function, and subsequently protein evolution, has increased dramatically, many aspects remain unexplored.” (Soskine & Tawfik, 2010)
“The distribution of mutational effects — beyond single proteins: The fitness effects of mutations in a cellular, let alone organismal, context are much more complex than in isolated proteins such as TEM1 β-lactamase. Future aims include the examination of fitness effects under native conditions (such as the expression of a chromosomal gene from its endogenous promoter) and the investigation of how the fate of mutated variants is affected by various buffering mechanisms and by mechanisms of protein trafficking and clearance.” (Soskine & Tawfik, 2010)
“The distribution of mutational effects — is an organism a sum of its proteins? Surprisingly, on preliminary examination, organisms seem to be more sensitive to mutations than their component proteins. The overall similarity in the distributions of fitness effects of mutations for a single protein and an intact organism, in addition to the differences, demand further exploration.” (Soskine & Tawfik, 2010)
“Studies of natural protein divergence: Our knowledge of protein mutations and their effects comes primarily from the study of model cases. In only a few cases has the natural divergence of a protein (or its clinical divergence, as in TEM1) been subsequently studied in the laboratory. Studies of actual adaptations will provide an interesting and more conclusive picture of how new genes and proteins diverge. Potential study targets include adaptations towards man-made chemicals, such as pesticides and herbicides in bacteria, insectsor plants. Secondary metabolism in plants is another rich source of functional diversification.” (Soskine & Tawfik, 2010)
“Reconstructions of ancestors and their divergence paths: Reconstructing ancestral genes and proteins and the divergence paths that led to contemporary proteins is a powerful approach that can also be applied to examine the mutational paths and the effects of individual mutations on the divergence process.” (Soskine & Tawfik, 2010)
“Compartmental adaptations: ‘Function’ refers not only to what a protein does in a living organism but also to where it does it. The same activity (for example, catalysing a given enzymatic transformation) can take place in different compartments or organisms. This means that proteins can be placed under different regulation schemes and be processed differently with regard to synthesis, transport, pH optimum, stability, and so on. It will be interesting to explore the sequence changes that drive compartmental adaptation, as these could be more intense than the sequence alterations that drive changes in activity itself.” (Soskine & Tawfik, 2010)
“Divergence by horizontal gene transfer: Horizontal (lateral) gene transfer is a common source of evolutionary innovation. A related gene imported from another organism partly resembles a newly formed duplicate, so it will be worth investigating whether the above-discussed mechanisms, mutational effects and their trade-offs apply.” (Soskine & Tawfik, 2010)
“Evolutionary rates: The rates of evolution of proteins (the average number of amino acid exchanges per position, per generation) are widely distributed between organisms (viral proteins provide a clear example), as well as within the same organism. Many factors might be involved, but the effects of the protein’s structure and its response to mutations (the distribution of mutational effects) remain unclear.” (Soskine & Tawfik, 2010)
“Epistasis and protein evolution: Although it is beyond the scope of this Review, epistasis is an important factor in protein evolution. If the effect of a given mutation depends on whether another mutation (or mutations) is present, the likelihood and mechanism of divergence will be affected. For example, new-function mutations may not be fixed unless a stabilizing, compensatory mutation is already present. Conversely, a compensatory mutation can be neutral or even deleterious on its own but beneficial in combination with a destabilizing mutation. Exploring the mutations that underline various divergence paths may provide new insights regarding the role of epistasis in directing the mechanism of divergence.” (Soskine & Tawfik, 2010)
“Other RdDM components, including DRM2, ARGONAUTE 4 (AGO4) and Pol V, are needed for siRNA accumulation for a subset of loci; however, these proteins do not seem to be involved in the initial production of siRNAs and are proposed to reinforce siRNA biogenesis by an unknown mechanism.” (Law & Jacobsen, 2010)
“The precise relationship among Pol II, Pol V and Pol IV remains elusive, but these studies suggest they may be more intimately connected than previously thought.” (Law & Jacobsen, 2010)
“ DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1), a putative chromatin-remodelling factor, and DMS3, an RdDM component with similarity to structural maintenance of chromosome (SMC) proteins, are needed for NRPE1 (NUCLEAR RNA POLYMERASE E1)chromatin association and for accumulation of IGN transcripts, although how these components are targeted is unknown.” (Law & Jacobsen, 2010)
“As DNMT3A seems to be a non-processive DNA methyltransferase, the formation of an oligomer could help to explain the observed periodic pattern of DNA methylation. Whether oligomerization occurs in vivo remains unknown, but it is tempting to propose a model in which interactions between DNMT3L and unmethylated H3K4 tails, or possibly between DNMT3A and other histone modifications or histone methyltransferases, might target and set the register for oligomerization of tetramers consisting of DNMT3A and DNMT3L, resulting in an ~8–10 bp periodicity.” (Law & Jacobsen, 2010)
“It was postulated that siRNAs generated in the vegetative nucleus might travel to the sperm cells and reinforce silencing by an unknown mechanism.” (Law & Jacobsen, 2010)
“Early studies of mammalian piRNA populations revealed that, unlike in D. melanogaster, mammalian piRNAs were not enriched for repetitive regions of the genome, leaving it unclear whether mammalian piRNAs function to silence transposons.” (Law & Jacobsen, 2010)
“Increases in antisense transcripts in met1 mutants were found to be rare and uncorrelated with body methylated genes9. Therefore, the function of body methylation remains poorly understood.” (Law & Jacobsen, 2010)
“The mechanism through which LSH1 functions in DNA methylation remains unknown.” (Law & Jacobsen, 2010)
“Whether direct protein interactions between CMT3 (Chromomethylase-3)and SUVH4 (SUPPRESSOR OF VARIEGATION 3-9 HOMOLOGUE 4)occur and aid in maintaining CHG methylation in plants is unknown.” (Law & Jacobsen, 2010)
“In mammals, the resulting single-nucleotide gap is then acted on by DNA polymerase-β and DNA ligase IIIα to repair the DNA through the short-patch BER pathway. Homologues of these enzymes have not been identified in plants, raising the possibility that plants use enzymes that are involved in the long-patch BER (base excision repair) pathway.” (Law & Jacobsen, 2010)
“After reciprocal crosses between two A. thaliana ecotypes (A genetically distinct population within a widely spread species.), siRNAs from the resultant silique (An elongated seed capsule that is formed after fertilization.) tissue (whichcontains the developing embryos) were sequenced, andnearly all of the Pol IV-dependent siRNAs that could be uniquely distinguished between the two ecotypeswere maternal in origin. What causes these loci to be maternally imprinted, whether this imprinting requiresDME and what function this massive extent of imprintingserves remain unknown. One hypothesis is that suchmaternal imprinting would allow recognition of selffrom non-self and have a suppressive effect on hybrids (Offspring that are produced by crossing two different populations within a single species).” (Law & Jacobsen, 2010)
“The mechanism (or mechanisms) through which the DME/ROS1 glycosylases are targeted to specific loci to carry out DNA demethylation is (are) unknown. These A. thaliana glycosylases are quite different from most other glycosylases: they are much larger and contain two conserved domains of unknown function. Whether these domains are required for targeting demethylation remains unknown.” (Law & Jacobsen, 2010)
“Proteins that are orthologous to the DME/ROS1 family of glycosylases have not been identified in mammals, and the existence of other enzymes that can directly remove methylated cytosines is controversial.” (Law & Jacobsen, 2010)
“In vivo, the observed decrease in methylation at the CYP27B1(cytochrome p450 27B1) promoter can occur in the absence of DNA replication — which suggests an active mechanism — and is dependent on the presence of a catalytically active MBD4 protein (an HhH-GPD thymine glycosylase that is related to the A. thaliana DME/ROS1 family of glycosylases and that has active mammalian homologues) that contains the serine residues that are targeted for phosphorylation. These recent findings need to be confirmed, and whether such a mechanism for the direct removal of methylated cytosines could account for DNA demethylation on a larger scale remains unknown.” (Law & Jacobsen, 2010)
“Despite the significant advances in our understanding of DNA methylation pathways, several key questions remain, especially surrounding the issue of targeting. How DNA methyltransferases are targeted by siRNAs and piRNAs in plants and mammals, respectively, remains elusive. In terms of DNA demethylation, whether DME is specifically targeted to many sites throughout the genome during gametogenesis or whether it non-selectively removes methylation remains unclear. Similarly, whether demethylation by the other DME/ROS1 family members is specifically directed to certain loci or whether the observed methylation pattern simply reflects a balance between the RdDM and demethylation pathways requires further investigation. Gaining further insights into mammalian DNA demethylation pathways and understanding how demethylation is targeted will be key challenges for future research.” (Law & Jacobsen, 2010)
