Recombinant DNA formation in a cell-free system from Xenopus laevis eggs

A cell-free system is described which formed very high levels of recombinant DNA structures in 4 hr at 26°C. It consisted of a single fraction of a high speed supernatant prepared from an extract of unfertilized eggs of the frog Xenopus laevis. This fraction eluted at 0.16?0.18 M Tris homogenization buffer from a DEAE-cellulose column. When two partially homologous supercoiled DNA molecules of different contour lengths were incubated simultaneously in this system, high levels of heterologous figure eight DNA structures were formed and observed by electron microscopy. Subsequent cleavage of the newly formed figure eight structures with Bam HI and Eco RI restriction endonucleases gave rise to “α structures” and “χ structures.” The observed figure eight structures presumably represent the recombination intermediate predicted by the Holliday model for genetic recombination.     

The mechanism of formation of Cellulose-like microfibrils in a cell-free system from Acetobacter xylinum

The mechanism of formation of cellulose-like microfibrils by a non-soluble, particulate enzyme and uridine diphosphoglucose (UDPG) in a cell-free system from Acetobacter xylinum was studied by transmission electron microscopy and X-ray diffraction. The suspension of particles to which the enzyme is adsorbed is composed of whole, dense ovoids, 50–250 nm long when wet, of fragments of the ovoids, and amorphous substance. There is a typical unit membrane around each ovoid but initially there is no trace of fibrillar material in the suspension. When the suspension of particles is incubated with UDPG, linear wisps of fibrils are produced which associate rapidly to form longer and wider threads, especially in 0.01 M NaCl. There is no visible attachment of the wisps to the particles. After 20 min incubation, threads with the typical morphology of cellulose microfibrils are formed that later tend to become entangled in clumps. The microfibrils are insoluble in hot, aqueous, alkaline solutions and resistant to the action of trypsin, but may be degraded by glusulase. After treatment with 1 M NaOH at 100° C or with cold 18% NaOH they show an X-ray diffraction pattern which resembles that of Cellulose II from mercerized, authentic bacterial cellulose. Incorporation of radioactive glucose into the insoluble residue is enhanced by drying of the cellulose microfibrils before alkaline digestion and especially by the addition of a gross excess of carrier cellulose after incubation. In this system there is no evidence for participation of linear, axial, synthesizing sites on the cell wall of the bacterium or for ordered, organized granules in the assembly of the microfibrils. That is, cellulose-like microfibrils may be formed in a cell-free system without the action of any of the previously suggested cell organelles. In addition, these observations are consistent with a previously described notion of a transient, hydrated, nascent, bacterial cellulose microfibril. The possibility that cellulose microfibrils of green plants may be formed in the same way is considered.N.R.C.C. 18314     

Survey of extrachromosomal circular DNA derived from plant satellite repeats

Background  

Satellite repeats represent one of the most dynamic components of higher plant genomes, undergoing rapid evolutionary changes of their nucleotide sequences and abundance in a genome. However, the exact molecular mechanisms driving these changes and their eventual regulation are mostly unknown. It has been proposed that amplification and homogenization of satellite DNA could be facilitated by extrachromosomal circular DNA (eccDNA) molecules originated by recombination-based excision from satellite repeat arrays. While the models including eccDNA are attractive for their potential to explain rapid turnover of satellite DNA, the existence of satellite repeat-derived eccDNA has not yet been systematically studied in a wider range of plant genomes.     

Survey of plant short tandem DNA repeats

Length variations in simple sequence tandem repeats are being given increased attention in plant genetics. Some short tandem repeats (STRs) from a few plant species, mainly those at the dinucleotide level, have been demonstrated to show polymorphisms and Mendelian inheritance. In the study reported here a search for all of the possible STRs ranging from mononucleotide up to tetranucleotide repeats was carried out on EMBL and GenBank DNA sequence databases of 3026 kb nuclear DNA and 1268 kb organelle DNA in 54 and 28 plant species (plus algae), respectively. An extreme rareness of STRs (4 STRs in 1268 kb DNA) was detected in organelle compared with nuclear DNA sequences. In nuclear DNA sequences, (AT)_n sequences were the most abundant followed by (A)_n · (T)_n, (AG)_n · (CT)_n, (AAT)_n · (ATT)_n, (AAC)_n · (GTT), (AGC)_n · (GCT)_n, (AAG)_n · (CTT)_n, (AATT)_n · (TTAA)_n, (AAAT)_n · (ATTT)_n and (AC)_n · (GT)_n sequences. A total of 130 STRs were found, including 49 (AT)_n sequences in 31 species, giving an average of 1 STR every 23.3 kb and 1 (AT)_n STR every 62 kb. An abundance comparable to that for the dinucleotide repeat was observed for the tri- and tetranucleotide repeats together. On average, there was 1 STR every 64.6 kb DNA in monocotyledons versus 1 every 21.2 kb DNA in dicotyledons. The fraction of STRs that contained G-C basepairs increased as the G+C contents went up from dicotyledons, monocotyledons to algae. While STRs of mono-, di- and tetranucleotide repeats were all located in non coding regions, 57% of the trinucleotide STRs containing G-C basepairs resided in coding regions.     

A role for DNA in the formation of nuclear estradiol-receptor complex in a cell-free system

Destruction of the DNA component of the nuclear pellet fraction of rat uterus homogenates with DNase prevented the formation of “nuclear” estradiol-receptor complex upon subsequent incubation with supernatant fraction containing 8S cytoplasmic estrogen-receptor complex. Nuclear pellet pretreatment with RNase had no effect on the reaction. DNase did not appear to release any component capable of binding estradiol or the 8S estradiol-receptor complex.     

A novel cell-free protein synthesis system

An efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. Using the new energy source, 3-phosphoglycerate (3-PGA), protein synthesis continues beyond 2 h. In contrast, the reaction rate slowed down considerably within 30–45 min using a conventional energy source, phosphoenol pyruvate (PEP) under identical reaction conditions. This improvement results in the production of twice the amount of protein obtained with PEP as an energy source. We have also shown that Gam protein of phage lambda, an inhibitor of RecBCD (ExoV), protects linear PCR DNA templates from degradation in vitro. Furthermore, addition of purified Gam protein in extracts of Escherichia coli BL21 improves protein synthesis from PCR templates to a level comparable to plasmid DNA template. Therefore, combination of these improvements should be amenable to rapid expression of proteins in a high-throughput manner for proteomics and structural genomics applications.     

Nuclear formation in a Drosophila cell-free system

A cell-free preparation obtained from 0- to 5-h-old Drosophila melanogaster embryos induces chromatin decondensation and nuclear formation from demembranated Xenopus sperm. Newly formed nuclei have a peripheral lamina, a double membrane, and structures resembling pore complexes. Indirect immunofluorescence analyses demonstrate the association of Drosophila lamins and DNA topoisomerase II with newly assembled nuclei.     

Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution

Background

Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data.

Results

Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. We assumed that the most abundant tandem repeat is the centromere DNA, which was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution.

Conclusions

While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animal and plant genomes. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes.     

Two-step recruitment of RNA-directed DNA methylation to tandem repeats

Tandem repeat sequences are frequently associated with gene silencing phenomena. The Arabidopsis thaliana FWA gene contains two tandem repeats and is an efficient target for RNA-directed de novo DNA methylation when it is transformed into plants. We showed that the FWA tandem repeats are necessary and sufficient for de novo DNA methylation and that repeated character rather than intrinsic sequence is likely important. Endogenous FWA can adopt either of two stable epigenetic states: methylated and silenced or unmethylated and active. Surprisingly, we found small interfering RNAs (siRNAs) associated with FWA in both states. Despite this, only the methylated form of endogenous FWA could recruit further RNA-directed DNA methylation or cause efficient de novo methylation of transgenic FWA. This suggests that RNA-directed DNA methylation occurs in two steps: first, the initial recruitment of the siRNA-producing machinery, and second, siRNA-directed DNA methylation either in cis or in trans. The efficiency of this second step varies depending on the nature of the siRNA-producing locus, and at some loci, it may require pre-existing chromatin modifications such as DNA methylation itself. Enhancement of RNA-directed DNA methylation by pre-existing DNA methylation could create a self-reinforcing system to enhance the stability of silencing. Tandem repeats throughout the Arabidopsis genome produce siRNAs, suggesting that repeat acquisition may be a general mechanism for the evolution of gene silencing.     

Nucleotide sequence of circular DNA molecules homologous to the 240 bp tandem repeats of the intergenic spacer of Drosophila melanogaster ribosomal DNA

We have determined the nucleotide sequence of ten 240 bp repeated sequences of the DNA intergenic spacer present in circular DNA molecules purified from D melanogaster embryos. No significant difference was found with the sequence of the chromosomal units. This suggests that most of the circular molecules homologous to the 240 bp repeats are generated by homologous recombination between adjacent chromosomal units.     

Subrepeats result from regional DNA sequence conservation in tandem repeats in Chironomus telomeres

Repeat units, widespread in eukaryotic genomes, are often partially or entirely built up of subrepeats. Homogenization between whole repeat units arranged in tandem usually can best be understood as a result of unequal crossing over. Such a mechanism is less plausible for maintaining similarities between subrepeats within a repeat unit when present in a regular array. In Chironomus telomeres, large blocks of tandemly repeated approximately 350 base-pair units contain two or three pairs of subrepeats with high mutual identities, embedded in linker DNA, non-repetitive within the repeat unit. Measurements of evolutionary base changes in two closely related species, Chironomus tentans and Chironomus pallidivittatus, permit us to conclude that the subrepeat arrangement is best explained as a consequence of regional sequence conservation after an earlier duplication of an ancestral half-unit.     

The complete mitochondrial genome sequence of Euphausia pacifica (Malacostraca: Euphausiacea) reveals a novel gene order and unusual tandem repeats

Euphausiid krill are dominant organisms in the zooplankton population and play a central role in marine ecosystems. Euphausia pacifica (Malacostraca: Euphausiacea) is one of the most important and dominant crustaceans in the North Pacific Ocean. In this paper, we described the gene content, organization, and codon usage of the E. pacifica mitochondrial genome. The mitochondrial genome of E. pacifica is 16 898 bp in length and contains a standard set of 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. Translocation of three transfer RNAs (trnL(1), trnL(2), and trnW) was found in the E. pacifica mitochondrial genome when comparing with the pancrustacean ground pattern. The rate of K(a)/K(s) in 13 protein-coding genes among three krill is much less than 1, which indicates a strong purifying selection within this group. The largest noncoding region in the E. pacifica mitochondrial genome contains one section with tandem repeats (4.7 x 154 bp), which are the largest tandem repeats found in malacostracan mitochondrial genomes so far. All analyses based on nucleotide and amino acid data strongly support the monophyly of Stomatopoda, Penaeidae, Caridea, Brachyura, and Euphausiacea. The Bayesian analysis of nucleotide and amino acid datasets strongly supports the close relationship between Euphausiacea and Decapoda, which confirms traditional findings. The maximum likelihood analysis based on amino acid data strongly supports the close relationship between Euphausiacea and Penaeidae, which destroys the monophyly of Decapoda.     

Construction of tandem repeats of DNA fragments by a polymerase chain reaction-based method

We describe a new application of megaprimer polymerase chain reaction (PCR) for constructing a tandemly repeated DNA sequence using the drought responsive element (DRE) from Arabidopsis thaliana as an example. The key feature in the procedure was PCR primers with partial complementarity but differing melting temperatures (T(m)). The reverse primer had a higher T(m), a 3' end complementary to the DRE sequence and a 5' region complementary to the forward primer. The initial cycles of the PCR were conducted at a lower primer annealing temperature to generate products that served as megaprimers in the later cycles conducted at a higher temperature to prevent annealing of the forward primer. The region of overlap between the megaprimers was extended for generating products with a variable copy number (one to four copies) of tandem DRE sequence repeats (71?bp). The PCR product with four tandem repeats (4× DRE) was used as a template to generate tandem repeats with higher copies (copy number large than four) or demonstrated to bind DRE-binding protein in an yeast one-hybrid assay using promotorless reporter genes (HIS and lacZ). This PCR protocol has numerous applications for generating DNA fragments of repeated sequences.     

NO induced novel DNA lesions: formation mechanism.

2'-Deoxyguanosine was treated with NO/O2 mixture at pH 7.0-7.8, and as well as the known major products such as 2'-deoxyxanthosine and 2'-deoxyoxanosine, some unidentified products were detected by RP-HPLC. In the present study, one of them has been characterized as a novel lesion, N2-nitro-2'-deoxyguanosine by spectrometric and chromatographic analysis. The mechanism for the production is also discussed.     

Copy number of tandem direct repeats within the inverted repeats of Marek's disease virus DNA

We previously reported that DNA of the oncogenic strain BC-1 of Marek's disease virus serotype 1 (MDV1) contains three units of tandem direct repeats with 132 base pair (bp) repeats within the inverted repeats of the long regions of the MDV1 genome, whereas the attenuated, nononcogenic viral DNA contains multiple units of tandem direct repeats (Maotani et al., 1986). In the present study, the difference in the copy numbers of 132 bp repeats of oncogenic and nononcogenic MDV1 DNAs in other strains of MDV1 was investigated by Southern blot hybridization. The main copy numbers in different oncogenic MDV1 strains differed: those of BC-1, JM and highly oncogenic Md5 were 3, 5 to 12 and 2, respectively. The viral DNA population with two units of repeats was small, but detectable, in cells infected with either the oncogenic BC-1 or JM strain. The MDV1 DNA in various MD cell lines contained either two units or both two and three units of repeats. The significance of the copy number of repeats in oncogenicity of MDV1 is discussed.