Insertion site specificity of the transposon Tn3.

The Tn3-deletion method [Davies and Hutchison, Nucleic Acids Res. 19, 5731-5738, (1991)] was used to sequence a 9.4 kb DNA fragment. Transpositional 'warm' spots were not a limiting factor but a 935 bp 'cold' spot was completed using a synthetic oligonucleotide primer. Two hundred and twenty three miniTn3 insertion sites from three sequencing projects were aligned and a 19 bp asymmetric consensus site was identified. There is no absolute sequence requirement at any position in this consensus, so insertion occurs promiscuously (approximately 37% of sites are potential targets). In our sequencing projects, multiply targeted sites always closely matched the consensus, although not all close matches were targeted frequently. The 935 bp cold spot showed no unusual features when analysed with the consensus sequence. The consensus can be used to accurately predict likely insertion sites in a new sequence. Synthetic oligonucleotides based on the consensus and a known hot spot for Tn3 were mutagenised. These sequences were not hot spots in our vectors, suggesting that the primary sequence alone is not sufficient to create an insertional hot spot. We conclude that some other factor, such as DNA secondary structure, also plays an important role in target site selection for the transposon Tn3.     

Identification of a New Genomic Hot Spot of Evolutionary Diversification of Protein Function

Establishment of phylogenetic relationships remains a challenging task because it is based on computational analysis of genomic hot spots that display species-specific sequence variations. Here, we identify a species-specific thymine-to-guanine sequence variation in the Glrb gene which gives rise to species-specific splice donor sites in the Glrb genes of mouse and bushbaby. The resulting splice insert in the receptor for the inhibitory neurotransmitter glycine (GlyR) conveys synaptic receptor clustering and specific association with a particular synaptic plasticity-related splice variant of the postsynaptic scaffold protein gephyrin. This study identifies a new genomic hot spot which contributes to phylogenetic diversification of protein function and advances our understanding of phylogenetic relationships.     

Principal causes of hot spots for cytosine to thymine mutations at sites of cytosine methylation in growing cells. A model, its experimental support and implications.

In Escherichia coli and human cells, many sites of cytosine methylation in DNA are hot spots for C to T mutations. It is generally believed that T.G mismatches created by the hydrolytic deamination of 5-methylcytosines (5meC) are intermediates in the mutagenic pathway. A number of hypotheses have been proposed regarding the source of the mispaired thymine and how the cells deal with the mispairs. We have constructed a genetic reversion assay that utilizes a gene on a mini-F to compare the frequency of occurrence of C to T mutations in different genetic backgrounds in exponentially growing E. coli. The results identify at least two causes for the hot spot at a 5meC: (1) the higher rate of deamination of 5meC compared to C generates more T.G than uracil.G (U.G) mismatches, and (2) inefficient repair of T.G mismatches by the very short-patch (VSP) repair system compared to the repair of U. G mismatches by the uracil-DNA glycosylase (Ung). This combination of increased DNA damage when the cytosines are methylated coupled with the relative inefficiency in the post-replicative repair of T.G mismatches can be quantitatively modeled to explain the occurrence of the hot spot at 5meC. This model has implications for mutational hot and cold spots in all organisms.     

Natural meiotic recombination hot spots in the Schizosaccharomyces pombe genome successfully predicted from the simple sequence motif M26

The M26 hot spot of meiotic recombination in Schizosaccharomyces pombe is the eukaryotic hot spot most thoroughly investigated at the nucleotide level. The minimum sequence required for M26 activity was previously determined to be 5'-ATGACGT-3'. Originally identified by a mutant allele, ade6-M26, the M26 heptamer sequence occurs in the wild-type S. pombe genome approximately 300 times, but it has been unclear whether any of these are active hot spots. Recently, we showed that the M26 heptamer forms part of a larger consensus sequence, which is significantly more active than the heptamer alone. We used this expanded sequence as a guide to identify a smaller number of sites most likely to be active hot spots. Ten of the 15 sites tested showed meiotic DNA breaks, a hallmark of recombination hot spots, within 1 kb of the M26 sequence. Among those 10 sites, one occurred within a gene, cds1(+), and hot spot activity of this site was confirmed genetically. These results are, to our knowledge, the first demonstration in any organism of a simple, defined nucleotide sequence accurately predicting the locations of natural meiotic recombination hot spots. M26 may be the first example among a diverse group of simple sequences that determine the distribution, and hence predictability, of meiotic recombination hot spots in eukaryotic genomes.     

A highly polymorphic meiotic recombination mouse hot spot exhibits incomplete repair

The recent mapping of recombination hot spots in the human genome has demonstrated that crossover is a nonrandom process that occurs at well-defined positions along chromosomes. However, the mechanisms that direct hot-spot turnover in complex mammalian genomes are poorly understood. Analyses of the human genome are impaired by the inability to genetically dissect and molecularly manipulate recombinogenic regions to test their roles in regulating hot spots. Here, using the BXD recombinant inbred strains as a crossover library, three new recombination hot spots have been identified on mouse chromosome 19. Analyses of a highly polymorphic recombination hot spot (HS22) revealed that approximately 4% of recombinant molecules display complex and incomplete repair with discontinuous conversion tracts, as well as persistent heteroduplex DNA at crossover sites in mature spermatozoa. Also, sequence analysis of the wild house mouse revealed instability at the center of this hot spot. This suggests that complete repair is not required for completion of mammalian meiosis, a scenario that leaves duplex DNA containing mismatches at crossover sites.     

Silencing Homologous RNA Recombination Hot Spots with GC-Rich Sequences in Brome Mosaic Virus

It has been observed that AU-rich sequences form homologous recombination hot spots in brome mosaic virus (BMV), a tripartite positive-stranded RNA virus of plants (P. D. Nagy and J. J. Bujarski, J. Virol. 71:3799–3810, 1997). To study the effect of GC-rich sequences on the recombination hot spots, we inserted 30-nucleotide-long GC-rich sequences downstream of AU-rich homologous recombination hot spot regions in parental BMV RNAs (RNA2 and RNA3). Although these insertions doubled the length of sequence identity in RNA2 and RNA3, the incidence of homologous RNA2 and RNA3 recombination was reduced markedly. Four different, both highly structured and nonstructured downstream GC-rich sequences had a similar “homologous recombination silencing” effect on the nearby hot spots. The GC-rich sequence-mediated recombination silencing mapped to RNA2, as it was observed when the GC-rich sequence was inserted at downstream locations in both RNA2 and RNA3 or only in the RNA2 component. On the contrary, when the downstream GC-rich sequence was present only in the RNA3 component, it increased the incidence of homologous recombination. In addition, upstream insertions of similar GC-rich sequences increased the incidence of homologous recombination within downstream hot spot regions. Overall, this study reveals the complex nature of homologous recombination in BMV, where sequences flanking the common hot spot regions affect recombination frequency. A replicase-driven template-switching model is presented to explain recombination silencing by GC-rich sequences.     

High-resolution recombination patterns in a region of human chromosome 21 measured by sperm typing

For decades, classical crossover studies and linkage disequilibrium (LD) analysis of genomic regions suggested that human meiotic crossovers may not be randomly distributed along chromosomes but are focused instead in "hot spots." Recent sperm typing studies provided data at very high resolution and accuracy that defined the physical limits of a number of hot spots. The data were also used to test whether patterns of LD can predict hot spot locations. These sperm typing studies focused on several small regions of the genome already known or suspected of containing a hot spot based on the presence of LD breakdown or previous experimental evidence of hot spot activity. Comparable data on target regions not specifically chosen using these two criteria is lacking but is needed to make an unbiased test of whether LD data alone can accurately predict active hot spots. We used sperm typing to estimate recombination in 17 almost contiguous ~5 kb intervals spanning 103 kb of human Chromosome 21. We found two intervals that contained new hot spots. The comparison of our data with recombination rates predicted by statistical analyses of LD showed that, overall, the two datasets corresponded well, except for one predicted hot spot that showed little crossing over. This study doubles the experimental data on recombination in men at the highest resolution and accuracy and supports the emerging genome-wide picture that recombination is localized in small regions separated by cold areas. Detailed study of one of the new hot spots revealed a sperm donor with a decrease in recombination intensity at the canonical recombination site but an increase in crossover activity nearby. This unique finding suggests that the position and intensity of hot spots may evolve by means of a concerted mechanism that maintains the overall recombination intensity in the region.     

Spatial analysis of 3' phosphoinositide signaling in living fibroblasts, III: influence of cell morphology and morphological Polarity

Activation of phosphoinositide (PI) 3-kinase is a required signaling pathway in fibroblast migration directed by platelet-derived growth factor. The pattern of 3' PI lipids in the plasma membrane, integrating local PI 3-kinase activity as well as 3' PI diffusion and turnover, influences the spatiotemporal regulation of the cytoskeleton. In fibroblasts stimulated uniformly with platelet-derived growth factor, visualized using total internal reflection fluorescence microscopy, we consistently observed localized regions with significantly higher or lower 3' PI levels than adjacent regions (hot and cold spots, respectively). A typical cell contained multiple hot spots, coinciding with apparent leading edge structures, and at most one cold spot at the rear. Using a framework for finite-element modeling with actual cell contact area geometries, we find that although the 3' PI pattern is affected by irregular contact area shape, cell morphology alone cannot explain the presence of hot or cold spots. Our results and analysis instead suggest that these regions reflect different local 3' PI dynamics, specifically through a combination of mechanisms: enhanced PI 3-kinase activity, reduced 3' PI turnover, and possibly slow/constrained 3' PI diffusion. The morphological polarity of the cell may thus bias 3' PI signaling to promote persistent migration in fibroblasts.     

Analysis of spontaneous base substitutions generated in mismatch-repair-deficient strains of Escherichia coli.

We used the lacI system of Escherichia coli to examine the distribution of base substitution mutations occurring spontaneously in different mismatch-repair-deficient strains. The examination of almost 1,200 nonsense mutations generated in strains carrying the mutS, mutH, and mutU alleles confirmed that transitions are highly favored over transversions. The detailed analysis of relative mutation rates at different sites revealed that the pattern of hot spots and cold spots is strikingly similar in each of the three strain backgrounds, strongly supporting the notions that the products of the three genes are part of the same system and that in the absence of any of the components the entire system fails to function. The distribution of mutations occurring in the absence of mismatch repair defined a pronounced topography of the lacI gene. There was no obvious correlation of the hot spots or cold spots with either nearest-neighbor sequences or A X T richness of the immediate surrounding sequence.     

Small-world network approach to identify key residues in protein-protein interaction

We show that protein complexes can be represented as small-world networks, exhibiting a relatively small number of highly central amino-acid residues occurring frequently at protein-protein interfaces. We further base our analysis on a set of different biological examples of protein-protein interactions with experimentally validated hot spots, and show that 83% of these predicted highly central residues, which are conserved in sequence alignments and nonexposed to the solvent in the protein complex, correspond to or are in direct contact with an experimentally annotated hot spot. The remaining 17% show a general tendency to be close to an annotated hot spot. On the other hand, although there is no available experimental information on their contribution to the binding free energy, detailed analysis of their properties shows that they are good candidates for being hot spots. Thus, highly central residues have a clear tendency to be located in regions that include hot spots. We also show that some of the central residues in the protein complex interfaces are central in the monomeric structures before dimerization and that possible information relating to hot spots of binding free energy could be obtained from the unbound structures.     

Cold spots of human mitochondrial DNA hypervariable segment 1

The distribution of mutations in hypervariable segment 1 (HVS1) of mitochondrial DNA (mtDNA) was analyzed for more than 37000 individuals from various regions of the world. The results were used to estimate the intensity of mutation processes and the features of the cold spot distribution in mtDNA. Analysis of the structural-functional organization and variation of HVS1 made it possible to associate a lower variation with functionally important HVS1 regions. The distribution of CAT cold spots in secondary DNA structures revealed a lack of correlation between the cold spot location and the structural type of the mtDNA region.     

Genetic analysis of clear-plaque mutations induced in bacteriophage lambda by 9-aminoacridine

Clear-plaque mutations were induced in the cI and cII genes of λ by treating lysogenic cells with 9-aminoacridine (9AA). Mapping of the mutations revealed that there were two hot spots for 9AA mutagenesis in cI, and one strong hot spot in cII. The hot spots in cI mapped close to 1 of the 3 runs of 4 G/C base-pairs and near the only run of 5 G/Cs, respectively, in this gene. Of 36 cI mutations tested, at most one mapped near a run of 6 A/T base-pairs. By analogy, the sequence responsible for the strong hot spot in cII may be the run of 6 G/Cs in this gene.     

Integration specificity of the hobo element of Drosophila melanogaster is dependent on sequences flanking the integration site

We analyzed the integration specificity of the hobo transposable element of Drosophila melanogaster. Our results indicate that hobo is similar to other transposable elements in that it can integrate into a large number of sites, but that some sites are preferred over others, with a few sites acting as integration hot spots. A comparison of DNA sequences from 112 hobo integration sites identified a consensus sequence of NTNNNNAC, but this consensus was insufficient to account for the observed integration specificity. To begin to define the parameters affecting hobo integration preferences, we analyzed sequences flanking a donor hobo element, as well as sequences flanking a hobo integration hot spot for their relative influence on hobo integration specificity. We demonstrate experimentally that sequences flanking a hobo donor element do not influence subsequent integration site preference, whereas, sequences contained within 31 base pairs flanking an integration hot spot have a significant effect on the frequency of integration into that site. However, sequence analysis of the DNA flanking several hot spots failed to identify any common sequence motif shared by these sites. This lack of primary sequence information suggests that higher order DNA structural characteristics of the DNA and/or chromatin may influence integration site selection by the hobo element. This revised version was published online in July 2006 with corrections to the Cover Date.     

How can we distinguish between mutational "hot spots" and "old sites" in human mtDNA samples?

New research into variation in mutation rates across nucleotide positions in human mitochondrial DNA (mtDNA) calls into question population genetics models that assume a constant mutation rate for all sites in a sequence, particularly for hypervariable control region segments I and II. Related to this research is discovering the extent to which highly polymorphic sites are really mutational "hot spots" rather than "old" sites rooted early in the phylogenetic tree. This issue is addressed through the analysis of linkage disequilibrium patterns in the mtDNAs of 10 human populations. Hot spots can be expected to show little or no disequilibrium since they can be interpreted as having randomly expressed patterns. In fact, the results suggest that many highly polymorphic sites are not old sites, but instead are hot spots. Suspected hot spots are listed and compared with hypervariable sites given by Wakeley (1993) and Hasegawa et al. (1993).     

Protein-protein interactions; coupling of structurally conserved residues and of hot spots across interfaces. Implications for docking

Hot spot residues contribute dominantly to protein-protein interactions. Statistically, conserved residues correlate with hot spots, and their occurrence can distinguish between binding sites and the remainder of the protein surface. The hot spot and conservation analyses have been carried out on one side of the interface. Here, we show that both experimental hot spots and conserved residues tend to couple across two-chain interfaces. Intriguingly, the local packing density around both hot spots and conserved residues is higher than expected. We further observe a correlation between local packing density and experimental deltadeltaG. Favorable conserved pairs include Gly coupled with aromatics, charged and polar residues, as well as aromatic residue coupling. Remarkably, charged residue couples are underrepresented. Overall, protein-protein interactions appear to consist of regions of high and low packing density, with the hot spots organized in the former. The high local packing density in binding interfaces is reminiscent of protein cores.     

基于ESDA的西北太平洋柔鱼资源空间热点区域及其变动研究

以西北太平洋(150°E—160°E、38°N—45°N)柔鱼Ommastrephes bartramii为研究对象,以2007和2010年鱿钓渔业的原始点位数据为基础,利用常规统计和探索性空间数据分析(ESDA)中的全局空间自相关分析方法,对西北太平洋柔鱼资源空间分布及其变动进行了研究。结果表明,西北太平洋柔鱼资源呈现较强的聚集分布特征,但不同空间位置差异较大。利用GIS和ESDA的局部自相关方法进行了柔鱼资源热冷点空间分布的制图,结果显示2007年研究区存在3个显著的热点和1个冷点,2010年与之存在较为明显的差异,具有1个热点和4个冷点。变化检测分析表明,两个年份间出现1个热点区域和1个冷点区域未发生变化,另有2个区域从2007年热点变成2010年冷点;此外,非热冷点之间的变动在研究区占据了主导地位。景观指数分析表明,研究区柔鱼资源热冷点格局的复杂性和自相似性并不高,但其异质性和聚集性非常强,且整体性和凝聚度均很高。分析认为,2007年西北太平洋柔鱼渔区的形成受温度和海流的影响,近一半作业渔区产量相对较高,但并未充分保证空间热点的大面积形成,空间热点和冷点分布面积大致相当;2010年整个西北太平洋柔鱼渔场受亲潮势力影响,空间热点较为集中、空间冷点较多且分散。     

In vitro antibody evolution targeting germline hot spots to increase activity of an anti-CD22 immunotoxin

Recombinant immunotoxin BL22, containing the Fv portion of an anti-CD22 antibody, produced complete remissions in most patients with drug-resistant hairy cell leukemia but had less activity in leukemias with low CD22 expression. Complementarity-determining region (CDR) mutagenesis is used to increase antibody affinity but can be difficult to perform successfully. We previously showed that antibodies with increased affinity and immunotoxins with increased activity could be obtained by directing mutations at specific DNA residues called hot spots. Because hot spots can arise either by somatic mutation or be present in the germline, we examined which type of hot spot is preferred for increasing antibody affinity. Initially, a second generation antibody phage-display library targeting a germline hot spot (Ser(30)-Asn(31)) within CDR1 of the antibody light chain was mutated. Substitution of serine 30 or asparagine 31 with arginine produced mutant immunotoxins with an affinity (0.8 nM) increased 7-fold over BL22 (5.8 nM) and 3-fold over the first generation mutant HA22 (2.3 nM). More importantly, a 10-fold increase in activity over BL22 and a 2-3-fold increase over HA22 were observed in various B lymphoma cell lines including WSU-CLL that contains only 5500 CD22 sites per cell. For comparison, two phage-display libraries targeting non-germline hot spots in heavy chain CDR1 and CDR3 were generated but did not produce Fv with increased affinity. Our results demonstrate that germline hot spots but not non-germline hot spots are effective for in vitro antibody affinity maturation.     

Hot Spots, Cold Spots, and the Geographic Mosaic Theory of Coevolution

Species interactions commonly coevolve as complex geographic mosaics of populations shaped by differences in local selection and gene flow. We use a haploid matching-alleles model for coevolution to evaluate how a pair of species coevolves when fitness interactions are reciprocal in some locations ("hot spots") but not in others ("cold spots"). Our analyses consider mutualistic and antagonistic interspecific interactions and a variety of gene flow patterns between hot and cold spots. We found that hot and cold spots together with gene flow influence coevolutionary dynamics in four important ways. First, hot spots need not be ubiquitous to have a global influence on evolution, although rare hot spots will not have a disproportionate impact unless selection is relatively strong there. Second, asymmetries in gene flow can influence local adaptation, sometimes creating stable equilibria at which species experience minimal fitness in hot spots and maximal fitness in cold spots, or vice versa. Third, asymmetries in gene flow are no more important than asymmetries in population regulation for determining the maintenance of local polymorphisms through coevolution. Fourth, intraspecific allele frequency differences among hot and cold spot populations evolve under some, but not all, conditions. That is, selection mosaics are indeed capable of producing spatially variable coevolutionary outcomes across the landscapes over which species interact. Altogether, our analyses indicate that coevolutionary trajectories can be strongly shaped by the geographic distribution of coevolutionary hot and cold spots, and by the pattern of gene flow among populations.