Cleavage of DNA.RNA hybrids by type II restriction enzymes.

The action of a number of restriction enzymes on DNA.RNA hybrids has been examined using hybrids synthesised with RNAs of cucumber mosaic virus as templates. The enzymes EcoRI, HindII, SalI, MspI, HhaI, AluI, TaqI and HaeIII cleaved the DNA strand of the hybrids (and possible also the RNA strand) into specific fragments. For four of these enzymes, HhaI, AluI, TaqI and HaeIII, comparison of the restriction fragments produced with the known sequences of the viral RNAs confirmed that they were recognising and cleaving the DNA strand of the hybrids at their correct recognition sequences. It is likely that the ability to utilise DNA.RNA hybrids as substrates is a general property of Type II restriction enzymes.     

Genetic transformation of Saccharomyces cerevisiae with single-stranded circular DNA vectors

We asked if single-stranded vector DNA molecules could be used to reintroduce cloned DNA sequences into a eukaryotic cell and cause genetic transformation typical of that observed using double-stranded DNA vectors. DNA was presented to Saccharomyces cerevisiae following a standard transformation protocol, genetic transformants were isolated, and the physical state of the transforming DNA sequence was determined. We found that single-stranded DNA molecules transformed yeast cells 10- to 30-fold more efficiently than double-stranded molecules of identical sequence. More cells were competent for transformation by the single-stranded molecules. Single-stranded circular (ssc) DNA molecules carrying the yeast 2 μ plasmid-replicator sequence were converted to autonomously replicating double-stranded circular (dsc) molecules, suggesting their efficient utilization as templates for DNA synthesis in the cell. Single-stranded DNA molecules carrying 2 μ plasmid non-replicator sequences recombined with the endogenous multicopy 2 μ plasmid DNA. This recombination yielded either the simple molecular adduct expected from homologous recombination (40% of the transformants examined) or aberrant recombination products carrying incomplete transforming DNA sequences, endogenous 2 μ plasmid DNA sequences, or both (60% of the transformants examined). These aberrant recombination products suggest the frequent use of a recombination pathway that trims one or both of the substrate DNA molecules. Similar aberrant recombination products were detected in 30% of the transformants in cotransformation experiments employing single-stranded and double-stranded DNA molecules, one carrying the 2 μ plasmid replicator sequence and the other the selectable genetic marker. We conclude that single-stranded DNA molecules are useful vectors for the genetic transformation of a eukaryotic cell. They offer the advantage of high transformation efficiency, and yield the same intracellular DNA species obtained upon transformation with double-stranded DNA molecules. In addition, single-stranded DNA molecules can participate in a recombination pathway that trims one or both DNA recombination substrates, a pathway not detected, at least at the same frequency, when transforming with double-stranded DNA molecules     

双单杂交技术选育亮盖灵芝与白肉灵芝优良性状杂交子

以亮盖灵芝Ganoderma lucidum和白肉灵芝G. leucocontextum为亲本,采用亮盖灵芝异核体双核菌丝与通过原生质体单核化获得的白肉灵芝单核体进行双单杂交,最终获得34株杂交子。采用ISSR技术对34株杂交子及亲本进行DNA多态性聚类分析,杂交子主要分为两个类群。对杂交子进行生长速度和灵芝酸含量对比,筛选到4株优质杂交子。进一步进行杂交子及亲本间HPLC指纹图谱对比,发现4株杂交子中有3株HPLC指纹图谱在具备两个亲本特征灵芝酸的同时,还有低极性灵芝酸的产生。     

Experimental Hybridization Reveals Biased Inheritance of the Internal Transcribed Spacer of the Nuclear Ribosomal DNA in Begonia ×taipeiensis

Begonia × taipeiensis C.-I Peng, a naturally occurring hybrid resulting from B. formosana × B. aptera, in Taiwan. To understand the inheritance of ribosomal DNA in unidirectional hybridization, experiments were conducted using B. formosana and B. aptera as ovule and pollen donors, respectively. The internal transcribed spacer region (ITS) of nuclear ribosomal DNA was amplified from the artificial hybrids, parental species, and natural hybrids. In contrast to the single type of ITS in the parental species, multiple sequences were cloned from both natural and artificial hybrids. A split decomposition network based on ITS nucleotide variation revealed that all but one (clone B14) of the hybrid sequences were “phylogenetically” closely related to B. formosana. Apparently, in such unidirectional hybridization, maternal DNA provided most of phylogenetic information. In the hybrid sequences, in addition to additive polymorphisms inherited from maternal (38.1%) and paternal (30.1%) plants, a novel nucleotide composition (31.8%) was also detected. The “new” characters are seen as noise in phylogenetic inference. They were probably obtained via intramolecular recombination, as gene conversion was not detected. The occurrence of genetic recombination appeared to be nonrandom, with a higher frequency in the ITS1 (3.14%) and ITS2 (3.42%) regions than in the 5.8S RNA gene (2.22%). Given the lack of sexual recombination in B. × taipeiensis and short time span, unequal crossing-over likely contributed to the heterogeneity of the ITS composition in the nuclear genome. Although the sterile hybrids have not attained their own lineage independent from the parental species, a high level of genetic diversity is transmitted asexually and maintained in these plants. Received 7 February 2001/ Accepted in revised form 31 May 2001     

A new approach to directed gene evolution by recombined extension on truncated templates (RETT)

We describe a new approach to in vitro DNA recombination technique termed recombined extension on truncated templates (RETT). RETT generates random recombinant gene library by template-switching of unidirectionally growing polynucleotides from primers in the presence of unidirectional single-stranded DNA fragments used as templates. RETT was applied to the recombination of two homologous chitinase genes from S. marcescens ATCC 21074 and S. liquefaciens GM1403. When the shuffled genes were examined by restriction mapping and sequence analysis, it was found that chimeric genes were produced at a high frequency (more than 70%) between two chitinase genes with 83% of sequence identity. The number of crossovers within each chimeric gene ranged from one to four, and the recombination points were randomly distributed along entire DNA sequence. We also applied RETT to directed evolution of a chitinase variant for enhancing thermostability. Chimeric chitinases that were more thermostable than the parental enzyme were successfully obtained by RETT-based recombination.     

Modes of reproduction of the hybridogenetic fish Squalius alburnoides in the Tejo and Guadiana rivers: an approach with microsatellites

The Squalius alburnoides complex was produced by hybridization between female S. pyrenaicus (PP genome) and an hypothetical paternal ancestor related with Anaecypris hispanica (AA genome). This study examined a diversity of mating types and found that there is the potential for considerable gene exchange among diploid, triploid and tetraploid hybrids. Using microsatellites, genomes were attributed to Squalius pyrenaicus (P) or reconstituted “nuclear non-hybrid” S. alburnoides (A), and subsequently confirmed in hybrids. Recombination of AA genomes in the “nuclear non-hybrid males” and recombination of the homogametic genomes (AA or PP) after exclusion of the heterogametic genome in triploid females (PAA) were observed by analysing parents and progeny of breeding experiments. Reproduction of tetraploids, generating a symmetric tetraploid genotype (PPAA) in the progeny, suggests a process that could potentially lead to the formation of a new bisexual species. Present results also support: (i) previously hypothesized pathways, in which PPA S. alburnoides females exclude the A genome, exhibit meiotic recombination between the P genomes and generate haploid eggs; (ii) reconstitution of the diploid maternal ancestor genome (PP) as well as of the unknown paternal ancestor (AA); (iii) the occurrence of the same genomic reproductive mechanisms when Anaecypris hispanica is involved; and (iv) the existence of an A. hispanica-like ancestor as the paternal ancestor of S. alburnoides.     

Retroviral integrase superfamily: the structural perspective

The retroviral integrase superfamily (RISF) comprises numerous important nucleic acid‐processing enzymes, including transposases, integrases and various nucleases. These enzymes are involved in a wide range of processes such as transposition, replication and repair of DNA, homologous recombination, and RNA‐mediated gene silencing. Two out of the four enzymes that are encoded by the human immunodeficiency virus—RNase H1 and integrase—are members of this superfamily. RISF enzymes act on various substrates, and yet show remarkable mechanistic and structural similarities. All share a common fold of the catalytic core and the active site, which is composed primarily of carboxylate residues. Here, I present RISF proteins from a structural perspective, describing the individual members and the common and divergent elements of their structures, as well as the mechanistic insights gained from the structures of RNase H1 enzyme complexes with RNA/DNA hybrids.     

Bacteriophage T4 ν mutants with a slow rate of thymine-dimer excision and a particular reactivation phenotype

Bacteriophage T4 uvs52 is a member of a class of UV-sensitive mutants with UV survival between T4 wild-type and v mutants. The mutation promotes recombination between extracellularly UV-irradiated phages. However, the location is adjacent to, or in, gene v. The question whether uvs52 is a v mutant with a particular type of v gene expression was investigated with acid solubilization of [¹⁴C]thymine dimers from DNA incubated with extracts from T4-infected cells. The dimer-removal activity of extracts from uvs52-infected cells was half that of wild-type T4, and similar to that of the v am5 and v op14 enzymes induced in the appropriate su⁺ hosts. The initial velocity of incision of UV-irradiated DNA by partially purified extracts from cells infected with uvs52 was 15% of that of the wild-type. Excision activity was not disturbed in such extracts. Further evidence of the location of uvs52 in gene v followed from the negative results from complementation assays with mixtures of extracts from cells infected with uvs52, uvs21 (another member of this class) or v1.

The relation between initial incision activity and substrate concentration (UV-irradiated ¹⁴C-DNA) suggested that the uvs52 endonuclease V is mutant with a high affinity and a slow rate of thymine-dimer incision. The reactivation phenotype was explained by assuming a slow rate of dimer excision in vivo as well, continuing throughout the reproductive cycle of the phage and leading to intermediate UV sensitivity and photoreactivability. The increased recombination frequencies were explained by assuming that the single-stranded regions of the DNA produced by incisions made at the end of the reproductive cycle are readily recombined into the growing DNA pool.     

An Examination of the Effects of Double-Strand Breaks on Extrachromosomal Recombination in Mammalian Cells

We studied the effects of double-strand breaks on intramolecular extrachromosomal homologous recombination in mammalian cells. Pairs of defective herpes thymidine kinase (tk) sequences were introduced into mouse Ltk- cells on a DNA molecule that also contained a neo gene under control of the SV40 early promoter/enhancer. With the majority of the constructs used, gene conversions or double crossovers, but not single crossovers, were recoverable. DNA was linearized with various restriction enzymes prior to transfection. Recombination events producing a functional tk gene were monitored by selecting for tk-positive colonies. For double-strand breaks placed outside of the region of homology, maximal recombination frequencies were measured when a break placed the two tk sequences downstream from the SV40 early promoter/enhancer. We observed no relationship between recombination frequency and either the distance between a break and the tk sequences or the distance between the tk sequences. The quantitative effects of the breaks appeared to depend on the degree of homology between the tk sequences. We also observed that inverted repeats recombined as efficiently as direct repeats. The data indicated that the breaks influenced recombination indirectly, perhaps by affecting the binding of a factor(s) to the SV40 promoter region which in turn stimulated or inhibited recombination of the tk sequences. Taken together, we believe that our results provide strong evidence for the existence of a pathway for extrachromosomal homologous recombination in mammalian cells that is distinct from single-strand annealing. We discuss the possibility that intrachromosomal and extrachromosomal recombination have mechanisms in common.     

Intermediate fertile Triticum aestivum （＋）Agropyron elongatum somatic hybrids are generated by low doses of UV irradiation

We report the production and characterization of somatic hybrids between Triticum aestivum L. and Agropyron elongatum (Host) Nevishi (the synonym is Thinopyrum ponticum). Asymmetric protoplast fusion was performed between Agropyron elongatum protoplasts irradiated with a low UV dose and protoplasts of wheat taken from nonregenerable suspension cultures. More than 40 green plantlets were obtained from 15 regenerated clones and one of them produced seeds. The phenotypes of the hybrid plants and seeds were intermediate between wheat and Agropyron elongatum. All of the regenerated calli and plants were verified as intergeneric hybrids on the basis of morphological observation and analysis of isozyme, cytological, 5SrDNA spacer sequences and random amplified polymorphic DNA (RAPD). RFLP analysis of the mitochondrial genome revealed evidence of random segregation and recombination of mtDNA.     

A Drosophila minisatellite contains multiple Chi sequences

We demonstrate the existence of polymorphic DNA minisatellites in Drosophila mauritiana, a close relative of D. melanogaster. One of these sequences (minisatellite mD4.2) consists of 13 tandemly repeated monomers, 10 of which are 33 base pairs long. Each of the repeat monomers contains sequences identical or very similar to the Chi sequence (GCTGGTGG), a signal for recBCD-dependent recombination in Escherichia coli. Sequences hybridizing to the mD4.2 minisatellite are present in at least 20–25 genomic locations and exhibit substantial variability among different populations of three Drosophila species and two populations of the house fly, Musca domestica. Interpopulational variation is a result of length differences rather than restriction site polymorphisms and genetic crosses establish that the hybridizing restriction fragment patterns have an underlying genetic basis. The presence of these sequences in the genetically well known Drosophila species allows critical examination of processes that produce and maintain the remarkable variability associated with these genomic regions.     

Cloning of Herpes simplex virus 2 DNA fragments in a plasmid vector

DNA isolated from virions of Herpes simplex type 2 (HSV-2) strain 333 was digested with various restriction enzymes and joined to the EK2 plasmid vector pBR322. The viral DNA sequences present in the hybrids were analyzed by restriction enzyme mapping and hybridization to fragments of HSV-2 DNA. The collection of recombinant molecules represents approx. 75% of the HSV-2-genome. In most cases, the structure of the recombinants seemed identical to the organization of authentic fragments of HSV-2 DNA, however, a few hybrids contained rearrangements of viral and plasmid sequences.     

六妹羊肚菌PacBio基因组测序和DNA 6mA甲基化分析

羊肚菌属于子囊门真菌,是世界范围内最受欢迎的食用菌之一。本研究通过PacBio单分子实时测序技术对我国四川省成功栽培的六妹羊肚菌Morchella sextelata SCLS菌株进行全基因组测序,获得其高质量核基因组组装,大小为53.57Mb,重复序列含量为17.03%,包含42条重叠群（contigs）,重叠群N50高达1.82Mb,其中13条重叠群两端均含有端粒重复序列,为完整的染色体。通过链特异性RNA-seq测序和转录本拼接,并结合多种基因预测策略,预测到13 182个蛋白编码基因,包括267个碳水化合物活性酶,11个次生代谢产物合成基因簇。通过与内蒙古地区栽培的六妹羊肚菌菌株NZTD180501373基因组比较发现,六妹羊肚菌进化过程中可能发生过染色体重组事件,二者具有33 055个SNP和48 726个InDel位点差异,并且各自拥有超过6Mb的特有序列。此外,相比于梯棱羊肚菌M. importuna,六妹羊肚菌SCLS菌株中与逆转录转座酶有关的orthogroup发生了扩张,并且拥有5个成员数超过100的特有逆转录转座酶基因家族。对SCLS菌株中的DNA N⁶腺嘌呤甲基化（6mA）修饰进行了鉴定,发现SCLS菌株基因组中0.42%的腺嘌呤被6mA甲基化,其含量显著高于已报道的6种双核亚界（Dikarya）真菌。6mA甲基化位点在逆转录转座子上显著富集,表明六妹羊肚菌中6mA甲基化可能调控逆转录转座子的活性,这也是首次在真菌中报道6mA甲基化与转座子相关。     

DNA repair in reduced genome: the Mycoplasma model

The occurrence of bacteria with a reduced genome, such as that found in Mycoplasmas, raises the question as to which genes should be enough to guarantee the genomic stability indispensable for the maintenance of life. The aim of this work was to compare nine Mycoplasma genomes in regard to DNA repair genes. An in silico analysis was done using six Mycoplasma species, whose genomes are accessible at GenBank, and M. synoviae, and two strains of M. hyopneumoniae, whose genomes were recently sequenced by The Brazilian National Genome Project Consortium and Southern Genome Investigation Program (Brazil) respectively. Considering this reduced genome model, our comparative analysis suggests that the DNA integrity necessary for life can be primarily maintained by nucleotide excision repair (NER), which is the only complete repair pathway. Furthermore, some enzymes involved with base excision repair (BER) and recombination are also present and can complement the NER activity. The absence of RecR and RecO-like ORFs was observed only in M. genitalium and M. pneumoniae, which can be involved with the conservation of gene order observed between these two species. We also obtained phylogenetic evidence for the recent acquisition of the ogt gene in M. pulmonis and M. penetrans by a lateral transference event. In general, the presence or nonexistence of repair genes is shared by all species analyzed, suggesting that the loss of the majority of repair genes was an ancestral event, which occurred before the divergence of the Mycoplasma species.     

The genetic analysis of F1 hybrid larvae between female Trichinella spiralis and male Trichinella britovi

Hybrids between female Trichinella spiralis and male Trichinella britovi were constructed. Then, hybrid genotype was characterized by DNA markers including mitochondrial cytochrome c oxidase subunit I (CO I) gene, the gene encoding the 43-kDa excretory–secretory (ES) protein, and genomic DNA fragments specific for T. spiralis and T. britovi identified from random amplified polymorphism DNA (RAPD). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial CO I gene revealed that all hybrids carried a T. spiralis pattern. The same analysis of the gene encoding the 43-kDa ES protein showed that each hybrid carried both T. spiralis and T. britovi gene type simultaneously. In the analysis of genomic DNA using RAPD-derived PCR primers, some hybrids carried T. spiralis and T. britovi-specific RAPD markers, while others carried the RAPD marker of T. spiralis only.     

Interplasmid illegitimate recombination in Bacillus subtilis cells

The illegitimate recombination between S. aureus plasmids pE194 (or pGG20-the hybrid between pE194 and E. coli plasmid pBR322) and pBD17 (plasmid pUB110 without Hpa-II-C-fragment) in B. subtilis was studied. Plasmid cointegrates were generated with the frequency of 1-3.10(-8). Among the 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all the parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions has revealed that in 8 cases recombination occurred between short homologous regions (9-15 b.p.). One of the recombinants resulted from nonhomologous recombination. The similarity between nucleotide sequences of recombination sites of two types of contegrates and those used for pE194 integration into the B. subtilis chromosome (Bashkirov et al. 1987) was demonstrated. Possible mechanisms of illegitimate recombination are discussed.     

Somatic hybridization between Lycopersicon peruvianum and L. pennellii: Regenerating ability and antibiotic resistance as selection systems

Somatic hybrids were obtained between the reproductively-isolated tomato species Lycopersicon peruvianum and L. pennellii. Leaf protoplasts of the former species and protoplasts from cell suspension cultures of the latter were fused with polyethylene glycol. A double selection scheme for fusion products was used on the basis of regeneration ability in L. peruvianum and resistance to the antibiotic G418 (2-deoxystreptamine) in an L. pennellii cell line. One tetraploid and four hexaploid hybrids were obtained from this fusion. The hexaploids might have originated by fusion of two L. pennellii protoplasts and one L. peruvianum protoplast. The hybrids were identified on the basis of isozymes (loci Prx-1, Prx-2, Prx-4, Prx-6, Prx-7, Pgi-1 and putative locus Mdh-1), leaf, flower morphology and epidermal hairs. The expression of antibiotic resistance and regeneration ability in the hybrids indicate that these are dominant or codominant traits. The sterility and subvitality of the resulting hybrids questions the value of somatic hybridization as a useful breeding approach in Lycopersicon.     

RNA at DNA Double-Strand Breaks: The Challenge of Dealing with DNA:RNA Hybrids

RNA polymerase II is recruited to DNA double-strand breaks (DSBs), transcribes the sequences that flank the break and produces a novel RNA type that has been termed damage-induced long non-coding RNA (dilncRNA). DilncRNAs can be processed into short, miRNA-like molecules or degraded by different ribonucleases. They can also form double-stranded RNAs or DNA:RNA hybrids. The DNA:RNA hybrids formed at DSBs contribute to the recruitment of repair factors during the early steps of homologous recombination (HR) and, in this way, contribute to the accuracy of the DNA repair. However, if not resolved, the DNA:RNA hybrids are highly mutagenic and prevent the recruitment of later HR factors. Here recent discoveries about the synthesis, processing, and degradation of dilncRNAs are revised. The focus is on RNA clearance, a necessary step for the successful repair of DSBs and the aim is to reconcile contradictory findings on the effects of dilncRNAs and DNA:RNA hybrids in HR.     

DNA条形码在热带龙脑香科树种鉴定中的应用

龙脑香科植物是东南亚地区重要的热带木材来源树种, 对其开展DNA条形码评估在林业监管及森林资源保护等方面具有非常重要的实际应用价值。通过对龙脑香科植物样品进行rbcL、matK、trnL-trnF和ITS2四个片段的扩增和测序, 结合GenBank下载的数据, 共获得龙脑香科树种14属244种共计899条序列。通过比较4个片段的通用性、序列特征、种内和种间的遗传变异, 基于Best Match (BM)、Best Close Match (BCM)、相似性搜索算法(BLAST)和邻接树(NJ) 4种方法评估DNA条形码对于龙脑香科树种的鉴定能力。结果表明, ITS2在龙脑香科树种中鉴定效率最高, 通过优化的扩增体系能够从该科植物叶片中获得较高质量的ITS2片段; 叶绿体matK片段扩增和测序效率为100%, 且种内及种间遗传变异明显, 鉴定成功率高于其它叶绿体片段, 并据此提出ITS2和matK适合作为龙脑香科树种的DNA条形码。     

DNA条形码在热带龙脑香科树种鉴定中的应用

龙脑香科植物是东南亚地区重要的热带木材来源树种, 对其开展DNA条形码评估在林业监管及森林资源保护等方面具有非常重要的实际应用价值。通过对龙脑香科植物样品进行rbcL、matK、trnL-trnF和ITS2四个片段的扩增和测序, 结合GenBank下载的数据, 共获得龙脑香科树种14属244种共计899条序列。通过比较4个片段的通用性、序列特征、种内和种间的遗传变异, 基于Best Match (BM)、Best Close Match (BCM)、相似性搜索算法(BLAST)和邻接树(NJ) 4种方法评估DNA条形码对于龙脑香科树种的鉴定能力。结果表明, ITS2在龙脑香科树种中鉴定效率最高, 通过优化的扩增体系能够从该科植物叶片中获得较高质量的ITS2片段; 叶绿体matK片段扩增和测序效率为100%, 且种内及种间遗传变异明显, 鉴定成功率高于其它叶绿体片段, 并据此提出ITS2和matK适合作为龙脑香科树种的DNA条形码。