Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells.

Recombinase encoded by the R gene of pSR1 of Zygosaccharomyces rouxii mediates reciprocal recombination between two specific recombination sites (RSs) to induce excision or inversion of the DNA segment that is flanked by the RSs. We report here that site-specific recombination mediated by this system takes place effeciently in tobacco cells. To monitor the recombination events in tobacco cells, we have constructed two types of cryptic beta-glucuronidase reporter gene in such a way that recombination such as inversion of the construct or excision of the intervening sequence results in their expression. When these cryptic reporter constructs were transiently introduced together with the R gene by electroporation into protoplasts of tobacco cells, beta-glucuronidase activity was detected. The cryptic reporter genes, when stably resident in the chromosome of tobacco cells, were also activated by the R gene. Structural analyses of the genomic DNA isolated from these tobacco cells showed that the R protein did in fact catalyze precise recombination between two copies of RSs in tobacco cells, with resultant activation of the cryptic reporter genes. This observation provides the basis for development of a DNA technology whereby large regions of DNA can be manipulated in plant chromosomes. Potential uses of this recombination system are discussed.     

Silent genes in prokaryotes

Abstract DNA sequence analysis provides excellent evidence for the origin of new genes, encoding new enzyme specificities or isozymes, via gene-duplication. New genes which arise in this way are likely to have arisen via silent gene intermediates. Such 'silent' genes are conceptually distinct from 'cryptic' genes which may also be silent; whereas cryptic genes are thought to be retained due to periodic selection, silent genes would be expected to have only a transient existence in the genome. Only very few of the known inactive genes are possibly (and with varying degrees of likelihood) of the 'silent' type.     

SALMONELLA TYPHIMURIUM-ESCHERICHIA COLI Hybrids for the Tryptophan Region

Fifty-eight hybrids were analyzed for their phenotypic stability, presence and nature of cryptic trp alleles and by P22-mediated transduction to yield percent homologies. The hybrids fall into 5 distinguishable classes: a haploid class in which selected E. coli genes replace equivalent sites in the S. typhimurium chromosome; three merodiploid classes in which the selected E. coli genes are integrated at novel sites in the S. typhimurium chromosome-on the same transducing fragment as the female genes selected against, with or without cryptic damage to a nearby gene, or not on the same transducing fragment; and one class in which recombination has not taken place and the E. coli DNA is presumed to be an exogenote. The homology values are heterogeneous and do not permit an accurate determination of the relative frequency of incorporation of the integrated male genetic material. A further study of 20 hybrids indicates that genetic rearrangements can occur in the hybrids.     

DNA methylation and epigenetic inheritance

Mammalian cell lines silence genes at low frequency by the methylation of promoter sequences. These silent genes can be reactivated at high frequency by the demethylating agent 5-azacytidine (5-aza-CR). The inactive and active epigenetic states of such genes are stably inherited. A method for silencing genes is now available. It involves treatment of permeabilized cells with 5-methyl deoxycytidine triphosphate (5-methyl dCTP) which is incorporated into DNA. The methylation of promoter sequences has been confirmed using the bisulfite genomic sequencing procedure. Methylated oligonucleotides homologous to promoter sequences might be used to specifically target and silence given genes, but results so far have not been conclusive. Treatments that silence or reactivate genes by changing DNA methylation can be referred to as epimutagens, as distinct from mutagens that act by changing DNA sequences. The epimutagen 5-aza-CR reactivates genes but has little mutagenic activity, whereas standard mutagens (such as ethyl methane sulfonate and ultraviolet light) have little reactivation activity. Nevertheless, much more information is required about the effects of DNA-damaging agents in changing DNA methylation and gene activity and also about the role of epimutations in tumor progression.     

Effect of N-methyl-N-nitrosourea on avian erythrocyte nuclei reactivation.

Avian erythrocytes are terminally differentiated cells but they can be reactivated by fusion with actively metabolising cells. We have examined the effects of treating the erythrocytes with a carcinogenic methylating agent, N-methyl-N-Nitrosourea (MNU), on the process of reactivation of adult and embryonic nuclei. We have found that the rate of nuclear enlargement is slightly lower in nuclei from MNU-treated cells than from control cells and that there is a marked delay of about 24 h in the appearance of nucleoli in both adult and embryonic cells. This is not due to an effect of MNU on ribosomal (r)DNA: the number of rDNA genes appears to be similar in treated and control cells. Also, the number of rDNA genes appears to be similar in adult and embryonic cells and in unreactivated and reactivated embryonic nuclei: thus, differences in reactivation rate between adult and embryonic cells, observed by us and others, can not be attributed to a gross difference in their ribosomal DNA contents, and reappearance of nucleoli on reactivation can not be due to an amplification of rDNA (i.e., to recovery of such genes if lost on terminal differentiation). We suggest that MNU, although a monofunctional alkylating agent, may cause increased association--possibly cross-linkage--between DNA and protein in chromatin, thereby hindering access of host cell reactivating proteins, especially to the nucleolar regions.     

The Evolution of Latent Genes in Subdivided Populations

We define latent genes as phenotypically silent DNA sequences which may be reactivated by various genetic mechanisms. Of interest is how they and their functional counterparts can be maintained at high frequency in the face of mutation and selection pressure. We propose a two-deme, three-allele model incorporating viability selection, mutation and migration in haploid populations. It is shown that polymorphism for the three alleles can be easily maintained for a wide range of biologically meaningful parameter values. Computer simulations were employed to gain qualitative insight into the global dynamics of the system. It was found that the dynamics of the latent allele is closely correlated with that of the functional allele. In addition, bias in the migration rates can strengthen or weaken selective conditions for preservation of the functional and latent alleles.     

Evolution of developmental traits

The evolution of plant development can be studied in many different ways, each of which provides new insights into how plants have been modified over evolutionary time. DNA sequencing shows that most developmental genes are under purifying selection and that obvious adaptive change in proteins is rare. This may indicate that most change occurs in cis-regulatory sequences, that tests for detecting selection lack power, or both. Gene duplications are common and often correlate with divergence of function, as predicted by theory. Studies of gene expression illuminate similarities among structures in disparate plant groups and indicate that the same genes have been deployed repeatedly for similar developmental ends. Comparative functional studies remain uncommon, but promise to illuminate how changing proteins lead to changes in development. Precise characterization of phenotypes by studies of developmental morphology is beginning to occur in some taxonomic groups. The genetic variation necessary for morphological change must originate as allelic polymorphism within populations; such polymorphism has been identified in grasses and in sunflowers, although it is often cryptic.     

The Heritable Activation of Cryptic Suppressor-Mutator Elements by an Active Element

A weakly active maize Suppressor-mutator (Spm-omega) element is able to heritably activate cryptic Spm elements in the maize genome. The spontaneous activation frequency, which is 1-5 x 10(-5) in the present genetic background, increases by about 100-fold in the presence of an Spm-omega and remains an order of magnitude above the background level a generation after removal of the activating Spm-omega. Sectorial somatic reactivation of cryptic elements can be detected phenotypically in kernels. Selection of such kernels constitutes an efficient selection for plants with reactivated Spm elements. Analysis of the reactivation process reveals that it is gradual and proceeds through genetically metastable intermediates that exhibit different patterns of element expression during plant development. Newly reactivated elements tend to return to an inactive form. However, the probability that an element will remain in a heritably active state increases when the element is maintained in the presence of an active Spm element for several generations.     

细菌固有耐药的研究进展

人们以往大多只关注由敏感细菌通过基因水平转移和自发突变方式获得的耐药性,而忽略了细菌对某类抗生素天然耐药的重要特性,细菌的这种特性又被称为固有耐药。固有耐药由固有耐药基因决定,这类基因是指存在于某类细菌染色体上位置保守的与耐药相关的一类基因。近年来,对固有耐药基因的研究已经越来越受到重视。固有耐药基因的发现不仅可以为新药研制提供药物作用靶标,而且通过阻断病原菌固有耐药基因还可使以往对该类菌不起作用的抗生素药物重新焕发抗菌活性。此外,已有研究表明固有耐药基因能够被移动元件捕获进而可水平转移至其他细菌,因此通过监测固有耐药基因可以预测耐药菌的出现。本文对传统的细菌固有耐药机制包括细胞膜的低渗透性和多药外排泵系统,以及已知重要病原菌的转移酶和代谢相关酶的固有耐药机制进行了介绍。同时,进一步对隐性固有耐药基因的特性进行了阐释,最后探讨了固有耐药与获得性耐药的进化关系,指出固有耐药基因很可能是一些获得性耐药基因的来源。     

Ecological divergence promotes the evolution of cryptic reproductive isolation

Speciation can involve the evolution of 'cryptic' reproductive isolation that occurs after copulation but before hybrid offspring are produced. Because such cryptic barriers to gene exchange involve post-mating sexual interactions, analyses of their evolution have focused on sexual conflict or traditional sexual selection. Here, we show that ecological divergence between populations of herbivorous walking sticks is integral to the evolution of cryptic reproductive isolation. Low female fitness following between-population mating can reduce gene exchange between populations, thus acting as a form of cryptic isolation. Female walking sticks show reduced oviposition rate and lower lifetime fecundity following between-population versus within-population mating, but only for mating between populations using different host-plant species. Our results indicate that even inherently sexual forms of reproductive isolation can evolve as a by-product of ecological divergence and that post-mating sexual interactions do not necessarily evolve independently of the ecological environment.     

Mitogenomics reveals high synteny and long evolutionary histories of sympatric cryptic nematode species

Species with seemingly identical morphology but with distinct genetic differences are abundant in the marine environment and frequently co‐occur in the same habitat. Such cryptic species are typically delineated using a limited number of mitochondrial and/or nuclear marker genes, which do not yield information on gene order and gene content of the genomes under consideration. We used next‐generation sequencing to study the composition of the mitochondrial genomes of four sympatrically distributed cryptic species of the Litoditis marina species complex (PmI, PmII, PmIII, and PmIV). The ecology, biology, and natural occurrence of these four species are well known, but the evolutionary processes behind this cryptic speciation remain largely unknown. The gene order of the mitochondrial genomes of the four species was conserved, but differences in genome length, gene length, and codon usage were observed. The atp8 gene was lacking in all four species. Phylogenetic analyses confirm that PmI and PmIV are sister species and that PmIII diverged earliest. The most recent common ancestor of the four cryptic species was estimated to have diverged 16 MYA. Synonymous mutations outnumbered nonsynonymous changes in all protein‐encoding genes, with the Complex IV genes (coxI‐III) experiencing the strongest purifying selection. Our mitogenomic results show that morphologically similar species can have long evolutionary histories and that PmIII has several differences in genetic makeup compared to the three other species, which may explain why it is better adapted to higher temperatures than the other species.     

Histone Variant HTZ1 Shows Extensive Epistasis with,but Does Not Increase Robustness to,New Mutations

Biological systems produce phenotypes that appear to be robust to perturbation by mutations and environmental variation. Prior studies identified genes that, when impaired, reveal previously cryptic genetic variation. This result is typically interpreted as evidence that the disrupted gene normally increases robustness to mutations, as such robustness would allow cryptic variants to accumulate. However, revelation of cryptic genetic variation is not necessarily evidence that a mutationally robust state has been made less robust. Demonstrating a difference in robustness requires comparing the ability of each state (with the gene perturbed or intact) to suppress the effects of new mutations. Previous studies used strains in which the existing genetic variation had been filtered by selection. Here, we use mutation accumulation (MA) lines that have experienced minimal selection, to test the ability of histone H2A.Z (HTZ1) to increase robustness to mutations in the yeast Saccharomyces cerevisiae. HTZ1, a regulator of chromatin structure and gene expression, represents a class of genes implicated in mutational robustness. It had previously been shown to increase robustness of yeast cell morphology to fluctuations in the external or internal microenvironment. We measured morphological variation within and among 79 MA lines with and without HTZ1. Analysis of within-line variation confirms that HTZ1 increases microenvironmental robustness. Analysis of between-line variation shows the morphological effects of eliminating HTZ1 to be highly dependent on the line, which implies that HTZ1 interacts with mutations that have accumulated in the lines. However, lines without HTZ1 are, as a group, not more phenotypically diverse than lines with HTZ1 present. The presence of HTZ1, therefore, does not confer greater robustness to mutations than its absence. Our results provide experimental evidence that revelation of cryptic genetic variation cannot be assumed to be caused by loss of robustness, and therefore force reevaluation of prior claims based on that assumption.     

基于微生物共培养的隐性基因簇激活策略

微生物次级代谢产物是药物先导化合物的重要源泉之一。随着测序技术的迅猛发展,越来越多的微生物基因组得以测序完成。伴随着测序技术的进步,生物信息学也得到了快速发展。基因组序列分析发现,链霉菌和丝状真菌等微生物中存在大量的已知的或未知的次级代谢物生物合成基因簇(secondary metabolite-biosynthetic gene clusters,SM-BGCs)。然而,在实验室培养条件下大部分基因簇无法表达或表达量很低,导致难以发现这些基因簇所对应的代谢产物,人们将这类基因簇称为“隐性基因簇”或“沉默基因簇”。通过调节基因簇中特异调控基因或基因簇外全局性调控基因的表达,对代谢途径的定向改造,以及将基因簇导入异源宿主等策略,能够激活部分隐性基因簇的表达。通过激活隐性基因簇的表达,能够发现通过常规实验室培养无法获得的具有独特生物活性的新结构代谢产物,成为创新药物的重要来源之一。然而,这些基因簇激活策略都严重依赖于对特定菌株或宿主的遗传操作。近年来,通过模拟自然混合培养中微生物间相互作用,开发了通过混合特定微生物菌株在厌氧或好氧条件下激活隐性基因簇的方法,称之为共培养激活策略。这种策略不...     

Aberrant cryptic responsiveness of the pCAT 3- and pGL3-promoter reporter vectors

Transfection analyses are an informative method to assess the activity of specific promoter or enhancer elements in mammalian cells. Commercially available reporter vectors can be extremely useful investigative tools for such studies. This study reports that the pCAT 3- and pGL3-promoter vectors display cryptic responsiveness to androgens when they contain a DNA insert, while the empty vector, a commonly used negative control, is nonresponsive. Our studies initially aimed to characterize novel androgen-responsive DNA sequences in human genomic DNA through transactivational analyses. An isolated DNA fragment, designated ARC-3, contained three putative androgen response element "half-sites" and was androgen-responsive when cloned into the pCAT3-promoter vector. While we originally believed this to be a novel enhancer element, subsequent analyses of this clone revealed that this vector displays cryptic activity in the presence of an androgen. This was confirmed by cloning several unrelated DNA fragments that did not contain any known classic response elements into the pCAT3-promoter vector, all of which were found to be responsive. The empty vector (negative control) was again nonresponsive. The ARC-3 DNA fragment was also weakly responsive to stimulation when cloned into the pGL3-promotor vector, which is identical to the pCAT3-promoter vector, with the exception of an intron located 5' of the chloramphenicol acetyltransferase gene, and the reporter genes. This work demonstrates that both the pCAT3- and pGL3-promoter vectors are inappropriate to assess androgen-responsive enhancers and emphasizes the importance of the careful selection of reporter vectors and controls when conducting transactivational analysis.