Fingerprinting genomes by use of PCR with primers that encode protein motifs or contain sequences that regulate gene expression

PCR primers of arbitrary nucleotide sequence have identified DNA polymorphisms useful for genetic mapping in a large variety of organisms. Although technically very powerful, the use of arbitrary primers for genome mapping has the disadvantage of characterizing DNA sequences of unknown function. Thus, there is no reason to anticipate that DNA fragments amplified by use of arbitrary primers will be enriched for either transcribed or promoter sequences that may be conserved in evolution. For these reasons, we modified the arbitrarily primed PCR method by using oligonucleotide primers derived from conserved promoter elements and protein motifs. Twenty-nine of these primers were tested individually and in pairwise combinations for their ability to amplify genomic DNA from a variety of species including various inbred strains of laboratory mice and Mus spretus. Using recombinant inbred strains of mice, we determined the chromosomal location of 27 polymorphic fragments in the mouse genome. The results demonstrated that motif sequence-tagged PCR products are reliable markers for mapping the mouse genome and that motif primers can also be used for genomic fingerprinting of many divergent species.     

Comparative genomic analysis identifies an ADP-ribosylation factor-like gene as the cause of Bardet-Biedl syndrome (BBS3)

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, pleiotropic human disorder characterized by obesity, retinopathy, polydactyly, renal and cardiac malformations, learning disabilities, and hypogenitalism. Eight BBS loci have been mapped, and seven genes have been identified. BBS3 was previously mapped to chromosome 3 by linkage analysis in a large Israeli Bedouin kindred. The rarity of other families mapping to the BBS3 locus has made it difficult to narrow the disease interval sufficiently to identify the gene by positional cloning. We hypothesized that the genomes of model organisms that contained the orthologues to known BBS genes would also likely contain a BBS3 orthologue. Therefore, comparative genomic analysis was performed to prioritize BBS candidate genes for mutation screening. Known BBS proteins were compared with the translated genomes of model organisms to identify a subset of organisms in which these proteins were conserved. By including multiple organisms that have relatively small genome sizes in the analysis, the number of candidate genes was reduced, and a few genes mapping to the BBS3 interval emerged as the best candidates for this disorder. One of these genes, ADP-ribosylation factor-like 6 (ARL6), contains a homozygous stop mutation that segregates completely with the disease in the Bedouin kindred originally used to map the BBS3 locus, identifying this gene as the BBS3 gene. These data illustrate the power of comparative genomic analysis for the study of human disease and identifies a novel BBS gene.     

DNA Damage Checkpoint, Damage Repair, and Genome Stability

Genomic DNA is under constant attack from both endogenous and exogenous sources of DNA damaging agents. Without proper care, the ensuing DNA damages would lead to alteration of genomic structure thus affecting the faithful transmission of genetic information. During the process of evolution, organisms have acquired a series of mechanisms responding to and repairing DNA damage, thus assuring the maintenance of genome stability and faithful transmission of genetic information. DNA damage checkpoint is one such important mechanism by which, in the face of DNA damage, a cell can respond to amplified damage signals, either by actively halting the cell cycle until it ensures that critical processes such as DNA replication or mitosis are complete or by initiating apoptosis as a last resort. Over the last decade, complex hierarchical interactions between the key components like ATM/ATR in the checkpoint pathway and various other mediators, effectors including DNA damage repair proteins have begun to emerge. In the meantime, an intimate relationship between mechanisms of damage checkpoint pathway, DNA damage repair, and genome stability was also uncovered. Reviewed hereinare the recent findings on both the mechanisms of activation of checkpoint pathways and their coordination with DNA damage repair machinery as well as their effect on genomic integrity.     

Molecular parameters of genome instability: roles of fragile genes at common fragile sites

Common chromosome fragile sites occur at specific sequences within mammalian genomes that exhibit apparent single-stranded regions in mitotic chromosomes on exposure of cells to replication stress. Recent progress in the characterization of sequences, and more precise mapping of common fragile sites in mammalian and yeast genomes, has led to the exact placement of large common fragile regions straddling the borders of chromosomal G and R bands, with early and late replicating genomic regions, respectively, and could lead to breakthroughs in understanding the function of these evolutionarily conserved but highly recombinogenic chromosome elements. Deficiency of genes involved in DNA damage checkpoint responses, such as ATR, CHK1, HUS1 leads to increased frequency of fragile site instability. Some of these fragile sites, particularly FRA3B, encode genes that are themselves involved in the protection of cells from DNA damage through various mechanisms. Protection of mammalian genomes from accumulation of DNA damage in somatic cells is critical during development, puberty and during the reproductive lifespan, and occurs through mechanisms involving surveillance of the genome for damage, signals to the cell cycle machinery to stop cell cycle progression, signals to repair machinery to repair damage, signals to resume cycling or initiate apoptotic programs, depending on the extent of damage and repair. When genes involved in these processes are altered or deleted, cancer can occur. The tumor suppressor gene, FHIT at the FRA3B locus, and possibly other fragile genes, is a common target of damage and paradoxically encodes a protein with roles in protection from DNA damage.     

DNA损伤检验点与损伤修复及基因组稳定性

生物有机体基因组DNA经常会受到内源或外源因素的影响而导致结构发生变化,产生损伤;在长期进化过程中,有机体也相应形成了一系列应对与修复损伤DNA,并维持染色体基因组正常结构功能的机制。其中DNA损伤检验点（DNA damage checkpoint）就是在感应DNA损伤的基础上,对损伤感应信号进行转导,或引起细胞周期的暂停,从而使细胞有足够的时间对损伤DNA进行修复,或最终导致细胞发生凋亡。DNA损伤检验点信号转导途径是一个高度保守的信号感应过程,整个途径大致可以分为损伤感应、信号传递及信号效应3个组成部分。其中3-磷脂酰肌醇激酶家族类成员ATM（ataxia-telangiectasia mutated）和ATR（ataxia-telangiectasia and Rad3-related）活性的增加构成整个途径活化的第一步。它们通过激活下游的效应激酶,Chk2／Chk1,通过协同作用许多其他调控细胞周期、DNA复制、DNA损伤修复及细胞凋亡等过程的蛋白质因子来实现细胞对DNA损伤的高度协调反应。近十几年,随着此领域研究的不断深入,人们逐步揭示了DNA损伤检验点途径发生过程中,各种核心组分通过与不同调节因子、效应因子及DNA损伤修复蛋白间的复杂相互作用,以实现监测感应异常DNA结构并实施相应反应的机制;其中,检验点衔接因子（mediators）及染色质结构,尤其是核小体组蛋白的共价修饰在调控ATM／ATR活性,促进ATM／ATR与底物间的相互作用以及介导DNA损伤位点周围染色质区域上多蛋白复合物在时间与空间上的动态形成发挥着重要的作用。同时,人们也开始发现DNA损伤检验点途径与DNA损伤修复、基因组稳定性以及肿瘤发生等过程之间某些内在的联系。该反应途径在通过协调细胞针对DNA损伤做出各种反应的基础上,直接或间接地参与或调控DNA损伤修复过程,并与DNA损伤修复途径协同作用最终保证染色体基凶组结构的完整性,而检验点途径的改变,则会引起基因组不稳定的发生,包括从突变频率的提高到大范围的染色体重排,以及染色体数量的畸变。如：突变发生在肿瘤形成早期,会大大增加肿瘤发生的几率。文章将对DNA损伤检验点途径机制及其对DNA损伤修复、基因组稳定性影响的最新进展进行综述。     

UvrD helicase suppresses recombination and DNA damage-induced deletions

UvrD, a highly conserved helicase involved in mismatch repair, nucleotide excision repair (NER), and recombinational repair, plays a critical role in maintaining genomic stability and facilitating DNA lesion repair in many prokaryotic species. In this report, we focus on the UvrD homolog in Helicobacter pylori, a genetically diverse organism that lacks many known DNA repair proteins, including those involved in mismatch repair and recombinational repair, and that is noted for high levels of inter- and intragenomic recombination and mutation. H. pylori contains numerous DNA repeats in its compact genome and inhabits an environment rich in DNA-damaging agents that can lead to increased rearrangements between such repeats. We find that H. pylori UvrD functions to repair DNA damage and limit homologous recombination and DNA damage-induced genomic rearrangements between DNA repeats. Our results suggest that UvrD and other NER pathway proteins play a prominent role in maintaining genome integrity, especially after DNA damage; thus, NER may be especially critical in organisms such as H. pylori that face high-level genotoxic stress in vivo.     

Chk1 in the DNA damage response: conserved roles from yeasts to mammals

Chk1 is an evolutionarily conserved protein kinase that functions to ensure genomic integrity upon genotoxic stress. Studies to date have revealed striking similarities among Chk1 pathways of different organisms. In this review we discuss what is known about Chk1 activation and what downstream factors are regulated by Chk1 to counter replication blocks and DNA damage induced by UV, IR, and other genotoxic agents. Where applicable, we also compare the role of Chk1 with that of the Chk2 protein kinase in the checkpoint responses.     

Identification,analysis, and utilization of conserved ortholog set markers for comparative genomics in higher plants

We have screened a large tomato EST database against the Arabidopsis genomic sequence and report here the identification of a set of 1025 genes (referred to as a conserved ortholog set, or COS markers) that are single or low copy in both genomes (as determined by computational screens and DNA gel blot hybridization) and that have remained relatively stable in sequence since the early radiation of dicotyledonous plants. These genes were annotated, and a large portion could be assigned to putative functional categories associated with basic metabolic processes, such as energy-generating processes and the biosynthesis and degradation of cellular building blocks. We further demonstrate, through computational screens (e.g., against a Medicago truncatula database) and direct hybridization on genomic DNA of diverse plant species, that these COS markers also are conserved in the genomes of other plant families. Finally, we show that this gene set can be used for comparative mapping studies between highly divergent genomes such as those of tomato and Arabidopsis. This set of COS markers, identified computationally and experimentally, may further studies on comparative genomes and phylogenetics and elucidate the nature of genes conserved throughout plant evolution.     

ABA in bryophytes: how a universal growth regulator in life became a plant hormone?

Abscisic acid (ABA) is not a plant-specific compound but one found in organisms across kingdoms from bacteria to animals, suggesting that it is a ubiquitous and versatile substance that can modulate physiological functions of various organisms. Recent studies have shown that plants developed an elegant system for ABA sensing and early signal transduction mechanisms to modulate responses to environmental stresses for survival in terrestrial conditions. ABA-induced increase in stress tolerance has been reported not only in vascular plants but also in non-vascular bryophytes. Since bryophytes are the key group of organisms in the context of plant evolution, clarification of their ABA-dependent processes is important for understanding evolutionary adaptation of land plants. Molecular approaches using Physcomitrella patens have revealed that ABA plays a role in dehydration stress tolerance in mosses, which comprise a major group of bryophytes. Furthermore, we recently reported that signaling machinery for ABA responses is also conserved in liverworts, representing the most basal members of extant land plant lineage. Conservation of the mechanism for ABA sensing and responses in angiosperms and basal land plants suggests that acquisition of this mechanism for stress tolerance in vegetative tissues was one of the critical evolutionary events for adaptation to the land. This review describes the role of ABA in basal land plants as well as non-land plant organisms and further elaborates on recent progress in molecular studies of model bryophytes by comparative and functional genomic approaches.     

Death and more: DNA damage response pathways in the nematode C. elegans

Genotoxic stress is a threat to our cells' genome integrity. Failure to repair DNA lesions properly after the induction of cell proliferation arrest can lead to mutations or large-scale genomic instability. Because such changes may have tumorigenic potential, damaged cells are often eliminated via apoptosis. Loss of this apoptotic response is actually one of the hallmarks of cancer. Towards the effort to elucidate the DNA damage-induced signaling steps leading to these biological events, an easily accessible model system is required, where the acquired knowledge can reveal the mechanisms underlying more complex organisms. Accumulating evidence coming from studies in Caenorhabditis elegans point to its usefulness as such. In the worm's germline, DNA damage can induce both cell cycle arrest and apoptosis, two responses that are spatially separated. The latter is a tightly controlled process that is genetically indistinguishable from developmental programmed cell death. Upstream of the central death machinery, components of the DNA damage signaling cascade lie and act either as sensors of the lesion or as transducers of the initial signal detected. This review summarizes the findings of several studies that specify the elements of the DNA damage-induced responses, as components of the cell cycle control machinery, the repairing process or the apoptotic outcome. The validity of C. elegans as a tool to further dissect the complex signaling network of these responses and the high potential for it to reveal important links to cancer and other genetic abnormalities are addressed.     

Comparative bacterial proteomics: analysis of the core genome concept

While comparative bacterial genomic studies commonly predict a set of genes indicative of common ancestry, experimental validation of the existence of this core genome requires extensive measurement and is typically not undertaken. Enabled by an extensive proteome database developed over six years, we have experimentally verified the expression of proteins predicted from genomic ortholog comparisons among 17 environmental and pathogenic bacteria. More exclusive relationships were observed among the expressed protein content of phenotypically related bacteria, which is indicative of the specific lifestyles associated with these organisms. Although genomic studies can establish relative orthologous relationships among a set of bacteria and propose a set of ancestral genes, our proteomics study establishes expressed lifestyle differences among conserved genes and proposes a set of expressed ancestral traits.     

Gene regulation in response to DNA damage

To deal with different kinds of DNA damages, there are a number of repair pathways that must be carefully orchestrated to guarantee genomic stability. Many proteins that play a role in DNA repair are involved in multiple pathways and need to be tightly regulated to conduct the functions required for efficient repair of different DNA damage types, such as double strand breaks or DNA crosslinks caused by radiation or genotoxins. While most of the factors involved in DNA repair are conserved throughout the different kingdoms, recent results have shown that the regulation of their expression is variable between different organisms. In the following paper, we give an overview of what is currently known about regulating factors and gene expression in response to DNA damage and put this knowledge in context with the different DNA repair pathways in plants. This article is part of a Special Issue entitled: Plant gene regulation in response to abiotic stress.     

A scaffold for the Chinese hamster genome

Chinese hamster ovary (CHO) cells are a prevalent tool in biological research and are among the most widely used host cell lines for production of recombinant therapeutic proteins. While research in other organisms has been revolutionized through the development of DNA sequence-based tools, the lack of comparable genomic resources for the Chinese hamster has impeded similar work in CHO cell lines. A comparative genomics approach, based upon the completely sequenced mouse genome, can facilitate genomic work in this important organism. Using chromosome synteny to define regions of conserved linkage between Chinese hamster and mouse chromosomes, a working scaffold for the Chinese hamster genome has been developed. Mapping CHO and Chinese hamster sequences to the mouse genome creates direct access to relevant information in public databases. Additionally, mapping gene expression data onto a chromosome scaffold affords the ability to interpret information in a genomic context, potentially revealing important structural and regulatory features in the Chinese hamster genome. Further development of this genomic scaffold will provide opportunities to use biomolecular tools for research in CHO cell lines today and will be an asset to future efforts to sequence the Chinese hamster genome.     

Comprehensive primer design for analysis of population genetics in non-sequenced organisms

Nuclear sequence markers are useful tool for the study of the history of populations and adaptation. However, it is not easy to obtain multiple nuclear primers for organisms with poor or no genomic sequence information. Here we used the genomes of organisms that have been fully sequenced to design comprehensive sets of primers to amplify polymorphic genomic fragments of multiple nuclear genes in non-sequenced organisms. First, we identified a large number of candidate polymorphic regions that were flanked on each side by conserved regions in the reference genomes. We then designed primers based on these conserved sequences and examined whether the primers could be used to amplify sequences in target species, montane brown frog (Rana ornativentris), anole lizard (Anolis sagrei), guppy (Poecilia reticulata), and fruit fly (Drosophila melanogaster), for population genetic analysis. We successfully obtained polymorphic markers for all target species studied. In addition, we found that sequence identities of the regions between the primer sites in the reference genomes affected the experimental success of DNA amplification and identification of polymorphic loci in the target genomes, and that exonic primers had a higher success rate than intronic primers in amplifying readable sequences. We conclude that this comparative genomic approach is a time- and cost-effective way to obtain polymorphic markers for non-sequenced organisms, and that it will contribute to the further development of evolutionary ecology and population genetics for non-sequenced organisms, aiding in the understanding of the genetic basis of adaptation.     

The G2-phase DNA-damage checkpoint

DNA damage causes cell-cycle delay before S phase, during replication and before mitosis. This involves a number of highly conserved proteins that sense DNA damage and signal the cell-cycle machinery. Kinases that were initially discovered in yeast model systems have recently been shown to regulate the regulators of cyclin-dependent kinases and to control the stability of p53. This shows the importance of checkpoint proteins for maintaining genome stability. Here, we discuss recent data from yeast and metazoans that suggest a remarkable conservation of the organization of the G2 DNA-damage checkpoint pathway.     

In the end, it's all structure

Chromosome end protection is essential for all organisms with linear genomes. Specialized structures, called telomeres, accomplish this protection by forming DNA-protein complexes that hide the natural chromosome ends from the DNA damage machinery. In mammalian cells protection takes place on several levels. Telomeric DNA forms large duplex loops with the help of telomeric proteins, consequently hiding the very tip of the telomere. Telomeric proteins play additional roles in protecting the end from degradation, regulating telomere length, and suppressing the DNA damage response machinery. Here we summarize the current knowledge about telomere structure, and discuss the future directions of the field.     

Isolation and chromosomal assignment of canine genomic BAC clones representing 25 cancer-related genes

An extensive number of genes have been implicated in the initiation and progression of human cancers, aiding our understanding of the genetic aetiology of this highly heterogeneous disease. In order to facilitate extrapolation of such information between species, we have isolated and physically mapped the canine orthologues of 25 well-characterised human cancer-related genes. The identity of PCR products representing each canine gene marker was first confirmed by DNA sequencing analysis. Each product was then radiolabelled and used to screen a genomic BAC library for the domestic dog. The chromosomal location of each positive clone in the canine karyotype was determined by fluorescence in situ hybridisation (FISH) onto canine metaphase preparations. Of the 25 genes, the FISH localisation of 21 correlated fully with that expected on the basis of known regions of conserved synteny between the human and canine genomes. Three correlated less closely, and the chromosomal location of the remaining marker showed no apparent correlation with current comparative mapping data. In addition to generating useful comparative mapping information, this panel of markers will act as a valuable resource for detailed study of candidate genes likely to be involved in tumourigenesis, and also forms the basis of a canine cancer-gene genomic microarray currently being developed for the study of unbalanced genomic aberrations in canine tumours.