Size Of Gene Specific Inverted Repeat - Dependent Gene Deletion In Saccharomyces cerevisiae

We describe here an approach for rapidly producing scar-free and precise gene deletions in S. cerevisiae with high efficiency. Preparation of the disruption gene cassette in this approach was simply performed by overlap extension-PCR of an invert repeat of a partial or complete sequence of the targeted gene with URA3. Integration of the prepared disruption gene cassette to the designated position of a target gene leads to the formation of a mutagenesis cassette within the yeast genome, which consists of a URA3 gene flanked by the targeted gene and its inverted repeat between two short identical direct repeats. The inherent instability of the inverted sequences in close proximity facilitates the self-excision of the entire mutagenesis cassette deposited in the genome and promotes homologous recombination resulting in a seamless deletion via a single transformation. This rapid assembly circumvents the difficulty during preparation of disruption gene cassettes composed of two inverted repeats of the URA3, which requires the engineering of unique restriction sites for subsequent digestion and T4 DNA ligation in vitro. We further identified that the excision of the entire mutagenesis cassette flanked by two DRs in the transformed S. cerevisiae is dependent on the length of the inverted repeat of which a minimum of 800 bp is required for effective gene deletion. The deletion efficiency improves with the increase of the inverted repeat till 1.2 kb. Finally, the use of gene-specific inverted repeats of target genes enables simultaneous gene deletions. The procedure has the potential for application on other yeast strains to achieve precise and efficient removal of gene sequences.     

Complete Sequence and Comparative Analysis of the Chloroplast Genome of Coconut Palm (Cocos nucifera)

Coconut, a member of the palm family (Arecaceae), is one of the most economically important trees used by mankind. Despite its diverse morphology, coconut is recognized taxonomically as only a single species (Cocos nucifera L.). There are two major coconut varieties, tall and dwarf, the latter of which displays traits resulting from selection by humans. We report here the complete chloroplast (cp) genome of a dwarf coconut plant, and describe the gene content and organization, inverted repeat fluctuations, repeated sequence structure, and occurrence of RNA editing. Phylogenetic relationships of monocots were inferred based on 47 chloroplast protein-coding genes. Potential nodes for events of gene duplication and pseudogenization related to inverted repeat fluctuation were mapped onto the tree using parsimony criteria. We compare our findings with those from other palm species for which complete cp genome sequences are available.     

Efficient transposition of IS204-derived plasmids in Streptomyces coelicolor

In order to study functional gene expression in Streptomyces coelicolor, a mini-transposon encoding the apramycin resistance gene aac(3)IV within its inverted repeat (IR) boundaries was constructed based on IS204, which was previously identified in the genome of Nocardia asteroides YP21. The mini-transposon and IS204 transposase gene were then put on a kanamycin-resistant conjugative plasmid pDZY101 that can only replicate in Escherichia coli. After mating with S. coelicolor A3(2) M145, resistant colonies arose efficiently on both apramycin and kanamycin plates. Plasmid rescue indicated that entire plasmids were inserted into the M145 genome with cleavage at an inverted repeat junction formed by the right inverted repeat (IRR) and the last 18 bp of the transposase gene, while the left inverted repeat (IRL) was untouched. Southern blot analysis of the mutants using an aac(3)IV gene probe showed that transposition of plasmid pDZY101 was genetically stable, with a single-copy insertion within the S. coelicolor M145 genome. Several mutagenesis libraries of S. coelicolor M145 were constructed using plasmid pDZY101 derivatives and the transposon insertion site was determined. The correlation between novel mutant phenotypes and previously uncharacterized genes was established and these transposon locations were widely scattered around the genome.     

Sequence analysis of the insertion element ISH1.8 and of associated structural changes in the genome of phage PhiH of the archaebacterium Halobacterium halobium

We have sequenced the insertion element ISH1.8 which can be present in one or two copies in the genome of phage ΦH of Halobacterium halobium. ISH1.8 is 1895 bp long, has no inverted repeat at its ends, and one only of the two copies is flanked by two 5-bp duplications. An 8-bp sequence composed of 4 bp from each end of ISH1.8 is present in both sites lacking the element. This 8-bp sequence could either be a specific insertion sequence or a part of the element that is left behind upon deletion. The plasmid pΦHL, consisting of the invertible L segment of the phage genome which is, in ΦH2 and ΦH5, flanked by two copies of ISH1.8, contains 112 bp of ISH1.8 and is released from the phage genome by recombination within a direct repeat of 9 bp. This 9-bp sequence (TCCCGCCCT) exists as an inverted repeat in ISH1.8 and therefore as two distinct repeats in phage genomes containing two copies of ISH1.8 in inverted orientation.     

Structural plasmid instability in Bacillus subtilis: Effect of direct and inverted repeats

Summary Using precise excision as a model system, we have quantified the effect of direct repeats, inverted repeats and the size of the spacer between the repeats in the process of deletion formation in Bacillus subtilis. Both in the presence and absence of inverted repeats, the frequency of precise excision was strongly dependent on the direct repeat length. By increasing the direct repeat length from 9 bp to 18 and 27 bp, the precise excision frequency was raised by 3 and 4 orders of magnitude, respectively. In addition, irrespective of the direct repeat length, the presence of flanking inverted repeats enhanced the excision frequency by 3 orders of magnitude. Varying the inverted repeat length and the spacer size over a wide range did not significantly affect the excision frequencies. These results fit well into a model for deletion formation by slipped mispairing during replication of single-stranded plasmid DNA.     

Complete Chloroplast Genome of Chionographis japonica (Willd.) Maxim. (Melanthiaceae): Comparative Genomics and Evaluation of Universal Primers for Liliales

The complete chloroplast genome of Chionographis japonica (Willd.) Maxim. (Melanthiaceae, Liliales) was mapped using polymerase chain reaction and the Sanger method. The circular double-stranded DNA was a typical quadripartite structure consisting of two inverted repeated regions (27,397 bp), a small single copy region (18,205 bp), and a large single-copy region (81,646 bp), with a total length of 154,645 bp. The genome consisted of 137 coding genes, including 91 protein-coding genes, 38 distinct tRNA, and 8 rRNA genes. The ycf15 and ycf68 genes had several internal stop codons interpreted as pseudogenes. The inverted repeat (IR) region expanded to part of the rps3 gene in the junction between large single-copy and IRA regions in C. japonica. We designed 785 primers, of which 481 were used to map the entire chloroplast genome of C. japonica. Primers were compared with the complete chloroplast sequence of Smilax china (Smilacaceae) to identify primers that could be used for other Liliales members and whole chloroplast genome sequencing. Of the primers used for C. japonica, 398 could be used with other smaller species within the order.     

Gene localization on the chloroplast DNA of the maiden hair fern;Adiantum capillus-veneris

Gene maps were constructed for the inverted repeat region and for the adjacent large single copy region of the chloroplast genome of the maiden hair fern,Adiantum capillus-veneris L. Gene order and organization was very different from the typical angiosperm chloroplast genome (e.g. tobacco). Elongation of inverted repeat and a minimum of two inversions must be postulated to account for the unusual genome structure.     

Mutants of Chlamydomonas reinhardtii with physical alterations in their chloroplast DNA

We have isolated nonphotosynthetic (acetate-requiring) mutants with physical alterations in chloroplast DNA following growth of haploid cells in the chloroplast specific mutagen 5-fluorodeoxyuridine (FdUrd) or treatment of FdUrd-grown diploid cells with X rays. About one-third of the nonphotosynthetic mutations resulting from FdUrd treatment alone show simple deletions. All eight of the mutants examined so far which were obtained with FdUrd plus X rays have deletions that are accompanied by rearrangements, including inversions or duplications. All the alterations extend into one of the two inverted repeat regions of the chloroplast genome which contain the ribosomal RNA cistrons. However, Southern hybridization experiments reveal that the rRNA cistrons are not deleted but instead are contained in new fragments. The relocated rRNA cistrons appear to be functional, since the mutants have normal levels of chloroplast ribosomes. In most cases the deletions and rearrangements are symmetrical and affect both inverted repeats in a similar fashion. An exception is the mutant ac-u-c-2–43, which lacks one inverted repeat region almost completely, including an entire set of rRNA genes. Three additional mutants, which fail to recombine with ac-u-c-2–43 to give photosynthetically competent cells, have smaller deletions in the same region of the genome. These physical mapping studies have allowed us to place the ac-u-c locus itself in a region of unique sequence DNA in a fragment, Ba10, which also includes the right-hand end of one inverted repeat.     

The First Complete Chloroplast Genome Sequences in Actinidiaceae: Genome Structure and Comparative Analysis

Actinidia chinensis is an important economic plant belonging to the basal lineage of the asterids. Availability of a complete Actinidia chloroplast genome sequence is crucial to understanding phylogenetic relationships among major lineages of angiosperms and facilitates kiwifruit genetic improvement. We report here the complete nucleotide sequences of the chloroplast genomes for Actinidia chinensis and A. chinensis var deliciosa obtained through de novo assembly of Illumina paired-end reads produced by total DNA sequencing. The total genome size ranges from 155,446 to 157,557 bp, with an inverted repeat (IR) of 24,013 to 24,391 bp, a large single copy region (LSC) of 87,984 to 88,337 bp and a small single copy region (SSC) of 20,332 to 20,336 bp. The genome encodes 113 different genes, including 79 unique protein-coding genes, 30 tRNA genes and 4 ribosomal RNA genes, with 16 duplicated in the inverted repeats, and a tRNA gene (trnfM-CAU) duplicated once in the LSC region. Comparisons of IR boundaries among four asterid species showed that IR/LSC borders were extended into the 5’ portion of the psbA gene and IR contraction occurred in Actinidia. The clap gene has been lost from the chloroplast genome in Actinidia, and may have been transferred to the nucleus during chloroplast evolution. Twenty-seven polymorphic simple sequence repeat (SSR) loci were identified in the Actinidia chloroplast genome. Maximum parsimony analyses of a 72-gene, 16 taxa angiosperm dataset strongly support the placement of Actinidiaceae in Ericales within the basal asterids.     

The Complete Plastid Genome of Lagerstroemia fauriei and Loss of rpl2 Intron from Lagerstroemia (Lythraceae)

Lagerstroemia (crape myrtle) is an important plant genus used in ornamental horticulture in temperate regions worldwide. As such, numerous hybrids have been developed. However, DNA sequence resources and genome information for Lagerstroemia are limited, hindering evolutionary inferences regarding interspecific relationships. We report the complete plastid genome of Lagerstroemia fauriei. To our knowledge, this is the first reported whole plastid genome within Lythraceae. This genome is 152,440 bp in length with 38% GC content and consists of two single-copy regions separated by a pair of 25,793 bp inverted repeats. The large single copy and the small single copy regions span 83,921 bp and 16,933 bp, respectively. The genome contains 129 genes, including 17 located in each inverted repeat. Phylogenetic analysis of genera sampled from Geraniaceae, Myrtaceae, and Onagraceae corroborated the sister relationship between Lythraceae and Onagraceae. The plastid genomes of L. fauriei and several other Lythraceae species lack the rpl2 intron, which indicating an early loss of this intron within the Lythraceae lineage. The plastid genome of L. fauriei provides a much needed genetic resource for further phylogenetic research in Lagerstroemia and Lythraceae. Highly variable markers were identified for application in phylogenetic, barcoding and conservation genetic applications.     

Inverted repeat sequences in the drosophila genome

The properties of inverted repeat (foldback) sequences in Drosophila melanogaster DNA have been studied by HAP chromatography and electron microscope methods. Electron microscope observations show that there is a broad distribution of lengths of the duplex regions of the inverted repeats from very short to greater than 15 kb, with number and weight average values of 1.35 kb and 5.0 kb respectively. About 20% of the inverted repeats are separated by a single-strand spacer with lengths too short to observe, but the other 80% have spacers, P, with lengths ranging from 0.5 kb to greater than 30 kb. The number average and weight average spacer lengths for the total sample are 2.7 kb and 6.1 kb. With respect to the lengths of the spacers, P, between inverted repeats, the Drosophila genome differs from that of most organisms which have been studied where the spacers P are mostly too short to be measured. EM and HAP studies suggest that the average center-to-center spacing between sets of inverted repeats is 40–80 kb. The HAP studies show that there is a broad range of thermal stabilities for the duplexes formed by reassociation of inverted repeat sequences. Kinetic analysis shows that all of the frequency components of the Drosophila genome are present in the inverted repeats, the loops P, and the flanking sequences. There is a somewhat larger proportion of middle repetitive DNA in those inverted repeat duplexes which are resistant to digestion by Mung Bean Endonuclease I. These enzyme resistant duplexes comprise about 3% of the entire genome. It is estimated that there are approximately 2000–4000 inverted repeat pairs in the entire genome.     

Stability of an inverted repeat in a human fibrosarcoma cell.

Deletions and rearrangements of DNA sequences within the genome of human cells result in mutations associated with human disease. We have developed a selection system involving a neo gene containing a DNA sequence inserted into the NcoI site that can be used to quantitatively assay deletion of this sequence from the chromosome. The spontaneous deletion from the neo gene of a 122 bp inverted repeat occurred at a rate of 2.1 x 10(-8) to <3.1 x 10(-9) revertants/cell/generation in three different cell lines. Deletion of the 122 bp inverted repeat occurred between 6 bp flanking direct repeats. Spontaneous deletion of a 122 bp non-palindromic DNA sequence flanked by direct repeats was not observed, indicating a rate of deletion of <3.1 x 10(-9) revertants/cell/generation. This result demonstrates that a 122 bp inverted repeat can exhibit a low level of instability in some locations in the chromosome of a human cell line.     

ORGANIZATION OF THE CHLOROPLAST GENOME OF THE FRESHWATER CENTRIC DIATOM CYCLOTELLA MENEGHINIANA1

We constructed a complete physical map and a partial gene map of the chloroplast genome of Cyclotella meneghiniana Kützing clone 1020-1a (Bacillariophyceae). The 128-kb circular molecule contains a 17-kb inverted repeat, which divides the genome into single copy regions of65 kb and 29 kb. This is the largest genome and inverted repeat found in any diatom examined to date. In addition to the 16S and 23S ribosomal RNA genes, the inverted repeat contains both the ndhD gene (as yet unexamined in other diatoms) and the psbA gene (located similarly in one of two other examined diatoms). The Cyclotella chloroplast genome exists as two equimolar populations of inversion isomers that differ in the relative orientation of their single copy sequences. This inversion heterogeneity presumably results from intramolecular recombination within the inverted repeat. For the first time, we map the ndhD, psaC, rpofi, rpoCl, and rpoC2 genes to the chloroplast genome of a chlorophyll c-containing alga. While the Cyclotella chloroplast genome retains some prokaryotic and land plant gene clusters and operons, it contains a highly rearranged gene order in the large and small single copy regions compared to all other examined diatom, algal, and land plant chloroplast genomes.     

An Integrated System for Precise Genome Modification in Escherichia coli

We describe an optimized system for the easy, effective, and precise modification of the Escherichia coli genome. Genome changes are introduced first through the integration of a 1.3 kbp Landing Pad consisting of a gene conferring resistance to tetracycline (tetA) or the ability to metabolize the sugar galactose (galK). The Landing Pad is then excised as a result of double-strand breaks by the homing endonuclease I-SceI, and replaced with DNA fragments bearing the desired change via λ-Red mediated homologous recombination. Repair of the double strand breaks and counterselection against the Landing Pad (using NiCl₂ for tetA or 2-deoxy-galactose for galK) allows the isolation of modified bacteria without the use of additional antibiotic selection. We demonstrate the power of this method to make a variety of genome modifications: the exact integration, without any extraneous sequence, of the lac operon (~6.5 kbp) to any desired location in the genome and without the integration of antibiotic markers; the scarless deletion of ribosomal rrn operons (~6 kbp) through either intrachromosomal or oligonucleotide recombination; and the in situ fusion of native genes to fluorescent reporter genes without additional perturbation.     

The chloroplast genome of a chlorophylla+c-containing alga,Odontella sinensis

The chloroplast genome of a marine centric diatom,Odontella sinensis, was cloned and sequenced. The circular genome is 119,704 bp in length (AC=Z67753;). It contains an inverted repeat sequence of 7,725 bp separating two single-copy regions of 38,908 and 65,346 bp, respectively, and 174 genes and open reading frames, of which nine are duplicated within the inverted repeat segments.     

A Simple and Effective Method for Construction of Escherichia coli Strains Proficient for Genome Engineering

Multiplex genome engineering is a standalone recombineering tool for large-scale programming and accelerated evolution of cells. However, this advanced genome engineering technique has been limited to use in selected bacterial strains. We developed a simple and effective strain-independent method for effective genome engineering in Escherichia coli. The method involves introducing a suicide plasmid carrying the λ Red recombination system into the mutS gene. The suicide plasmid can be excised from the chromosome via selection in the absence of antibiotics, thus allowing transient inactivation of the mismatch repair system during genome engineering. In addition, we developed another suicide plasmid that enables integration of large DNA fragments into the lacZ genomic locus. These features enable this system to be applied in the exploitation of the benefits of genome engineering in synthetic biology, as well as the metabolic engineering of different strains of E. coli.