Conditional chromosome splitting in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using the homing endonuclease PI-SceI

A novel chromosome engineering technology is described which enables conditional splitting of natural chromosomes in haploid cells of the yeast Saccharomyces cerevisiae. The technology consists of introduction of a recognition sequence for the homing endonuclease PI-SceI into the S. cerevisiae genome and conditional expression of the gene encoding the PI-SceI enzyme under the control of the MET3 promoter. To test the technology, we split chromosome V upstream of GLC7 by use of the autonomously replicating sequence (ARS)-added polymerase-chain-reaction-mediated chromosome-splitting (ARS-PCS) method that we recently developed. A recognition sequence for PI-SceI was subsequently introduced downstream of the GLC7 locus. Splitting was analyzed following induction of the PI-SceI-encoding gene. Approximately 50% of the clones tested had the expected minichromosome harboring only the GLC7 gene, suggesting that any desired chromosomal region may be converted into a new chromosome by use of this method. Because this technology allows initial construction of a strain harboring multiple constructs prior to subsequent induction of random chromosome loss events under specific selective conditions, we propose that this technology may be applicable to reconstructing the S. cerevisiae genome by means of combinatorial loss of minichromosomes.     

Advances in molecular methods to alter chromosomes and genome in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A fundamental issue in biotechnology is how to breed useful strains of microorganisms for efficient production of valuable biomaterials. On-going and more recent developments in gene manipulation technologies and chromosomal and genomic modifications in particular have facilitated important contributions in this area. “Chromosome manipulation technology” as an outgrowth of “gene manipulation technology” may provide opportunities for creating novel strains of organisms with a variety of genomic constitutions. A simple and rapid chromosome splitting technology called “PCR-mediated chromosome splitting” (PCS) that we recently developed has made it possible to manipulate chromosomes and genomes on a large scale in an industrially important microorganism, Saccharomyces cerevisiae. This paper focuses on recent advances in molecular methods for altering chromosomes and genome in S. cerevisiae featuring chromosome splitting technology. These advances in introducing large-scale genomic modifications are expected to accelerate the breeding of novel strains for biotechnological purposes, and to reveal functions of presently uncharacterized chromosomal regions in S. cerevisiae and other organisms.     

Chromosome structure: DNA nucleotide sequence elements of a subset of the minichromosomes of the protozoan Trypanosoma brucei.

The genome of the protozoan Trypanosoma brucei contains a set of about 100 minichromosomes of about 50 to 150 kb in size. The small size of these chromosomes, their involvement in antigenic variation, and their mitotic stability make them ideal candidates for a structural analysis of protozoan chromosomes and their telomeres. We show that a subset of the minichromosomes is composed predominantly of simple-sequence DNA, with over 90% of the length of the minichromosome consisting of a tandem array of 177-bp repeats, indicating that these molecules have limited protein-coding capacity. Proceeding from the tip of the telomere to a chromosome internal position, a subset of the minichromosomes contained the GGGTTA telomere repeat, a 29-bp telomere-derived repeat, a region containing 74-bp G + C-rich direct repeats separated by approximately 155 bp of A + T-rich DNA that has a bent character, and 50 to 150 kb of the 177-bp repeat. Several of the minichromosome-derived telomeres did not encode protein-coding genes, indicating that the repertoire of telomeric variant cell surface glycoprotein genes is restricted to some telomeres only. The telomere organization in trypanosomes shares striking similarities to the organization of telomeres and subtelomeres in humans, yeasts, and plasmodia. An electron microscopic analysis of the minichromosomes showed that they are linear molecules without abnormal structures in the main body of the chromosome. The structure of replicating molecules indicated that minichromosomes probably have a single bidirectional origin of replication located in the body of the chromosome. We propose a model for the structure of the trypanosome minichromosomes.     

Accumulation of multiple copies of maize minichromosomes

Multiple copies of B chromosomes in maize (Zea mays) can accumulate in the genome using the B chromosome's accumulation mechanism, specifically nondisjunction at the second pollen mitosis and preferential fertilization of the egg. Using this mechanism, we accumulated 4 different-sized minichromosomes derived from the B chromosome to test the chromosome limits of the cell. The accumulation of normal B chromosomes is associated with multiple phenotypes including white stripes and asymmetric leaf blades, but when minichromosomes are accumulated these symptoms are absent. We also found that multiple B chromosome-derived minichromosomes can coexist with A chromosome-derived minichromosomes. During the years that these experiments were conducted, we found many B chromosome rearrangements and fragments, 2 recoverable A chromosome fragments, and observed a minichromosome breakage-fusion-bridge cycle in roots.     

The African trypanosome genome

The haploid nuclear genome of the African trypanosome, Trypanosoma brucei, is about 35 Mb and varies in size among different trypanosome isolates by as much as 25%. The nuclear DNA of this diploid organism is distributed among three size classes of chromosomes: the megabase chromosomes of which there are at least 11 pairs ranging from 1 Mb to more than 6 Mb (numbered I-XI from smallest to largest); several intermediate chromosomes of 200-900 kb and uncertain ploidy; and about 100 linear minichromosomes of 50-150 kb. Size differences of as much as four-fold can occur, both between the two homologues of a megabase chromosome pair in a specific trypanosome isolate and among chromosome pairs in different isolates. The genomic DNA sequences determined to date indicated that about 50% of the genome is coding sequence. The chromosomal telomeres possess TTAGGG repeats and many, if not all, of the telomeres of the megabase and intermediate chromosomes are linked to expression sites for genes encoding variant surface glycoproteins (VSGs). The minichromosomes serve as repositories for VSG genes since some but not all of their telomeres are linked to unexpressed VSG genes. A gene discovery program, based on sequencing the ends of cloned genomic DNA fragments, has generated more than 20 Mb of discontinuous single-pass genomic sequence data during the past year, and the complete sequences of chromosomes I and II (about 1 Mb each) in T. brucei GUTat 10.1 are currently being determined. It is anticipated that the entire genomic sequence of this organism will be known in a few years. Analysis of a test microarray of 400 cDNAs and small random genomic DNA fragments probed with RNAs from two developmental stages of T. brucei demonstrates that the microarray technology can be used to identify batteries of genes differentially expressed during the various life cycle stages of this parasite.     

Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing

Rice is an important crop and a model system for monocot genomics, and is a target for whole genome sequencing by the International Rice Genome Sequencing Project (IRGSP). The IRGSP is using a clone by clone approach to sequence rice based on minimum tiles of BAC or PAC clones. For chromosomes 10 and 3 we are using an integrated physical map based on two fingerprinted and end-sequenced BAC libraries to identifying a minimum tiling path of clones. In this study we constructed and tested two rice genomic libraries with an average insert size of 10 kb (10-kb library) to support the gap closure and finishing phases of the rice genome sequencing project. The HaeIII library contains 166,752 clones covering approximately 4.6x rice genome equivalents with an average insert size of 10.5 kb. The Sau3AI library contains 138,960 clones covering 4.2x genome equivalents with an average insert size of 11.6 kb. Both libraries were gridded in duplicate onto 11 high-density filters in a 5 x 5 pattern to facilitate screening by hybridization. The libraries contain an unbiased coverage of the rice genome with less than 5% contamination by clones containing organelle DNA or no insert. An efficient method was developed, consisting of pooled overgo hybridization, the selection of 10-kb gap spanning clones using end sequences, transposon sequencing and utilization of in silico draft sequence, to close relatively small gaps between sequenced BAC clones. Using this method we were able to close a majority of the gaps (up to approximately 50 kb) identified during the finishing phase of chromosome-10 sequencing. This method represents a useful way to close clone gaps and thus to complete the entire rice genome.     

DNA repeat arrays in chicken and human genomes and the adaptive evolution of avian genome size

Background

Birds have smaller average genome sizes than other tetrapod classes, and it has been proposed that a relatively low frequency of repeating DNA is one factor in reduction of avian genome sizes.

Results

DNA repeat arrays in the sequenced portion of the chicken (Gallus gallus) autosomes were quantified and compared with those in human autosomes. In the chicken 10.3% of the genome was occupied by DNA repeats, in contrast to 44.9% in human. In the chicken, the percentage of a chromosome occupied by repeats was positively correlated with chromosome length, but even the largest chicken chromosomes had repeat densities much lower than those in human, indicating that avoidance of repeats in the chicken is not confined to minichromosomes. When 294 simple sequence repeat types shared between chicken and human genomes were compared, mean repeat array length and maximum repeat array length were significantly lower in the chicken than in human.

Conclusions

The fact that the chicken simple sequence repeat arrays were consistently smaller than arrays of the same type in human is evidence that the reduction in repeat array length in the chicken has involved numerous independent evolutionary events. This implies that reduction of DNA repeats in birds is the result of adaptive evolution. Reduction of DNA repeats on minichromosomes may be an adaptation to permit chiasma formation and alignment of small chromosomes. However, the fact that repeat array lengths are consistently reduced on the largest chicken chromosomes supports the hypothesis that other selective factors are at work, presumably related to the reduction of cell size and consequent advantages for the energetic demands of flight.     

Genomic segments cloning and analysis of Cotesia plutellae polydnavirus using plasmid capture system

Cotesia plutellae polydnaviruses (CpBV) has a segmented genome consisting of multiple circular double stranded DNAs. Recently, we have developed an easy, simple, and convenient system based on Tn7 transposition in order to clone genomic segments of CpBV in Escherichia coli cell and designated plasmid capture system (PCS). The PCS donor-S transferred a pUC19 origin of replication and an ampicillin resistance marker into CpBV genomic DNA by in vitro transposition. Through PCS system, we were able to clone 53 genomic clones ranging from 0.1 to 25.5 kb and further they were classified into 29 segments by their sizes and restriction endonuclease patterns. Among them, a complete nucleotide sequence of CpBV-S28 segment was determined and 10 putative genes were predicted from this segment. Interestingly, 9 of 10 putative ORFs had high level of similarities with catalytic domain of protein tyrosine phosphatase. Also, ORF2807 showed similarity with EP1-like proteins of C. congregata polydnavirus.     

Chimeric mitochondrial minichromosomes of the human body louse, Pediculus humanus: evidence for homologous and non-homologous recombination

The mitochondrial (mt) genome of the human body louse, Pediculus humanus, consists of 18 minichromosomes. Each minichromosome is 3 to 4 kb long and has 1 to 3 genes. There is unequivocal evidence for recombination between different mt minichromosomes in P. humanus. It is not known, however, how these minichromosomes recombine. Here, we report the discovery of eight chimeric mt minichromosomes in P. humanus. We classify these chimeric mt minichromosomes into two groups: Group I and Group II. Group I chimeric minichromosomes contain parts of two different protein-coding genes that are from different minichromosomes. The two parts of protein-coding genes in each Group I chimeric minichromosome are joined at a microhomologous nucleotide sequence; microhomologous nucleotide sequences are hallmarks of non-homologous recombination. Group II chimeric minichromosomes contain all of the genes and the non-coding regions of two different minichromosomes. The conserved sequence blocks in the non-coding regions of Group II chimeric minichromosomes resemble the "recombination repeats" in the non-coding regions of the mt genomes of higher plants. These repeats are essential to homologous recombination in higher plants. Our analyses of the nucleotide sequences of chimeric mt minichromosomes indicate both homologous and non-homologous recombination between minichromosomes in the mitochondria of the human body louse.     

Cytogenetic analysis of an asymmetric potato hybrid

An asymmetric potato hybrid and its parental lines were cytogenetically examined. DAPI (4'-6-diamidino-2-phenylindole) staining was used to count chromosomes in all analysed lines and revealed the presence of minichromosomes in the hybrid genome. Fluorescent in situ hybridization (FISH) with rDNA sequence as a probe helped to determine the ploidy level of analysed lines and revealed that none of the minichromosomes contains rDNA repeats. CMA (chromomycin A3) band occurred to be a new chromosome marker that identifies potato chromosome No.1. It was possible to detect a deletion in one of four chromosomes No. 1 of the asymmetric potato hybrid. On the basis of these analyses a karyotype of the asymmetric hybrid was constructed.     

Programmed Minichromosome Elimination as a Mechanism for Somatic Genome Reduction in Tetrahymena thermophila

The maintenance of chromosome integrity is crucial for genetic stability. However, programmed chromosome fragmentations are known to occur in many organisms, and in the ciliate Tetrahymena the five germline chromosomes are fragmented into hundreds of minichromosomes during somatic nuclear differentiation. Here, we showed that there are different fates of these minichromosomes after chromosome breakage. Among the 326 somatic minichromosomes identified using genomic data, 50 are selectively eliminated from the mature somatic genome. Interestingly, many and probably most of these minichromosomes are eliminated during the growth period between 6 and 20 doublings right after conjugation. Genes with potential conjugation-specific functions are found in these minichromosomes. This study revealed a new mode of programmed DNA elimination in ciliates similar to those observed in parasitic nematodes, which could play a role in developmental gene regulation.     

A versatile and general splitting technology for generating targeted YAC subclones

Yeast artificial chromosomes (YAC) splitting technology was developed as a means to subclone any desired region of eukaryotic chromosomes from one YAC into new YACs. In the present study, the conventional YAC splitting technology was improved by incorporating PCR-mediated chromosome splitting technique and by adding autonomously replicating sequence (ARS) to the system. To demonstrate the performance of the improved method, a 60-kb region from within a 590-kb YAC (clone CIC9e2 from Arabidopsis thaliana chromosome 5) that could not be subcloned using the original method was split to convert into a replicating YAC. Two template plasmids, pSK-KCA and pSKCLY, were used to generate two splitting fragments by PCR. Two splitting fragments consisted of telomeric (C₄A₂)₆ repeats, 400-bp target region, CEN4, H4ARS and Km^r (selective marker for plant transformants), or CgLEU2. These splitting fragments were introduced into Saccharomyces cerevisiae harboring the 100-kb split YAC generated by splitting of the 590-kb YAC and containing the 60-kb region. Among 12 Leu⁺ transformants, four exhibited the expected karyotype in which two newly split 40- and 60-kb chromosomes were generated. These results demonstrate that the improved method can convert a targeted region of a eukaryotic chromosome within a YAC into a replicating YAC.     

Minichromosome analysis of chromosome pairing, disjunction, and sister chromatid cohesion in maize

With the advent of engineered minichromosome technology in plants, an understanding of the properties of small chromosomes is desirable. Twenty-two minichromosomes of related origin but varying in size are described that provide a unique resource to study such behavior. Fourteen minichromosomes from this set could pair with each other in meiotic prophase at frequencies between 25 and 100%, but for the smaller chromosomes, the sister chromatids precociously separated in anaphase I. The other eight minichromosomes did not pair with themselves, and the sister chromatids divided equationally at meiosis I. In plants containing one minichromosome, the sister chromatids also separated at meiosis I. In anaphase II, the minichromosomes progressed to one pole or the other. The maize (Zea mays) Shugoshin protein, which has been hypothesized to protect centromere cohesion in meiosis I, is still present at anaphase I on minichromosomes that divide equationally. Also, there were no differences in the level of phosphorylation of Ser-10 of histone H3, a correlate of cohesion, in the minichromosomes in which sister chromatids separated during anaphase I compared with the normal chromosomes. These analyses suggest that meiotic centromeric cohesion is compromised in minichromosomes depending on their size and cannot be maintained by the mechanisms used by normal-sized chromosomes.     

Efficient combination of large DNA in vitro: in gel site specific recombination (IGSSR) of PAC fragments containing alpha satellite DNA and the human HPRT gene locus.

In an attempt to combine a cloned genomic copy of a selectable gene with different cloned centromeric sequences to develop mammalian artificial chromosomes (MAC) we used site specific recombination mediated by purified Cre recombinase acting on the loxP sequence in PAC vector DNA. A new method was required to purify highly concentrated, virtually 100% intact PAC DNA which could be stored for a long period. Here we show the efficient linking of linearized PACs containing alpha satellite DNA from chromosomes X and 17 with sizes of 125 and 140 kb, respectively, to a 95 kb restriction fragment derived from a 175 kb PAC containing the intact human HPRT gene locus.     

t-loops at trypanosome telomeres

Mammalian telomeres form large duplex loops (t-loops) that may sequester chromosome ends by invasion of the 3' TTAGGG overhang into the duplex TTAGGG repeat array. Here we document t-loops in Trypanosoma brucei, a kinetoplastid protozoan with abundant telomeres due to the presence of many minichromosomes. These telomeres contained 10-20 kb duplex TTAGGG repeats and a 3' TTAGGG overhang. Electron microscopy of psoralen/UV cross-linked DNA revealed t-loops in enriched telomeric restriction fragments and at the ends of isolated minichromosomes. In mammals, t-loops are large (up to 25 kb), often comprising most of the telomere. Despite similar telomere lengths, trypanosome t-loops were much smaller (approximately 1 kb), indicating that t-loop sizes are regulated. Coating of non-cross-linked minichromosomes with Escherichia coli single-strand binding protein (SSB) often revealed 3' overhangs at both telomeres and several cross-linked minichromosomes had t-loops at both ends. These results suggest that t-loops and their prerequisite 3' tails can be formed on the products of both leading and lagging strand synthesis. We conclude that t-loops are a conserved feature of eukaryotic telomeres.     

Meiotic transmission of an in vitro-assembled autonomous maize minichromosome

Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes. As a step toward improving transgenic crop production, we report the development of autonomous maize minichromosomes (MMCs). We constructed circular MMCs by combining DsRed and nptII marker genes with 7-190 kb of genomic maize DNA fragments containing satellites, retroelements, and/or other repeats commonly found in centromeres and using particle bombardment to deliver these constructs into embryogenic maize tissue. We selected transformed cells, regenerated plants, and propagated their progeny for multiple generations in the absence of selection. Fluorescent in situ hybridization and segregation analysis demonstrated that autonomous MMCs can be mitotically and meiotically maintained. The MMC described here showed meiotic segregation ratios approaching Mendelian inheritance: 93% transmission as a disome (100% expected), 39% transmission as a monosome crossed to wild type (50% expected), and 59% transmission in self crosses (75% expected). The fluorescent DsRed reporter gene on the MMC was expressed through four generations, and Southern blot analysis indicated the encoded genes were intact. This novel approach for plant transformation can facilitate crop biotechnology by (i) combining several trait genes on a single DNA fragment, (ii) arranging genes in a defined sequence context for more consistent gene expression, and (iii) providing an independent linkage group that can be rapidly introgressed into various germplasms.     

Dissecting large and complex genomes: flow sorting and BAC cloning of individual chromosomes from bread wheat

The analysis of the complex genome of common wheat (Triticum aestivum, 2n = 6x = 42, genome formula AABBDD) is hampered by its large size ( approximately 17 000 Mbp) and allohexaploid nature. In order to simplify its analysis, we developed a generic strategy for dissecting such large and complex genomes into individual chromosomes. Chromosome 3B was successfully sorted by flow cytometry and cloned into a bacterial artificial chromosome (BAC), using only 1.8 million chromosomes and an adapted protocol developed for this purpose. The BAC library (designated as TA-3B) consists of 67 968 clones with an average insert size of 103 kb. It represents 6.2 equivalents of chromosome 3B with 100% coverage and 90% specificity as confirmed by genetic markers. This method was validated using other chromosomes and its broad application and usefulness in facilitating wheat genome analysis were demonstrated by target characterization of the chromosome 3B structure through cytogenetic mapping. This report on the successful cloning of flow-sorted chromosomes into BACs marks the integration of flow cytogenetics and genomics and represents a great leap forward in genetics and genomic analysis.     

Linkage disequilibrium maps constructed with common SNPs are useful for first-pass disease association screens

To develop an efficient strategy for mapping genetic factors associated with common diseases, we constructed linkage disequilibrium (LD) maps of human chromosomes 5, 7, 17, and X. These maps consist of common single nucleotide polymorphisms at an average intermarker distance of 100 kb. The genotype data from these markers in a panel of American samples of European descent were analyzed to produce blocks of markers in strong pair-wise LD. Power calculations were used to guide block definitions and predicted that high-level LD maps would be useful in initial genome scans for susceptibility alleles in case-control association studies of complex diseases. As anticipated, LD blocks on the X chromosome were larger and covered more of the chromosome than those found on the autosomes.     

Fragmented mitochondrial genomes are present in both major clades of the blood-sucking lice (suborder Anoplura): evidence from two Hoplopleura rodent lice (family Hoplopleuridae)

Background

The suborder Anoplura contains 540 species of blood-sucking lice that parasitize over 840 species of eutherian mammals. Fragmented mitochondrial (mt) genomes have been found in the lice of humans, pigs, horses and rats from four families: Pediculidae, Pthiridae, Haematopinidae and Polyplacidae. These lice, eight species in total, are from the same major clade of the Anoplura. The mt genomes of these lice consist of 9–20 minichromosomes; each minichromosome is 1.5–4 kb in size and has 1–8 genes. To understand mt genome fragmentation in the other major clade of the Anoplura, we sequenced the mt genomes of two species of rodent lice in the genus Hoplopleura (family Hoplopleuridae).

Results

We identified 28 mt genes on 10 minichromosomes in the mouse louse, Ho. akanezumi; each minichromosome is 1.7–2.7 kb long and has 1–6 genes. We identified 34 mt genes on 11 minichromosomes in the rat louse, Ho. kitti; each minichromosome is 1.8–2.8 kb long and has 1–5 genes. Ho. akanezumi also has a chimeric minichromosome with parts of two rRNA genes and a full-length tRNA gene for tyrosine. These two rodent lice share the same pattern for the distribution of all of the protein-coding and rRNA genes but differ in tRNA gene content and gene arrangement in four minichromosomes. Like the four genera of blood-sucking lice that have been investigated in previous studies, the Hoplopleura species have four minichromosomes that are only found in this genus.

Conclusions

Our results indicate that fragmented mt genomes were present in the most recent common ancestor of the two major clades of the blood-sucking lice, which lived ~75 million years ago. Intra-genus variation in the pattern of mt genome fragmentation is common in the blood-sucking lice (suborder Anoplura) and genus-specific minichromosomes are potential synapomorphies. Future studies should expand into more species, genera and families of blood-sucking lice to explore further the phylogenetic utility of the novel features associated with fragmented mt genomes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-751) contains supplementary material, which is available to authorized users.     

Chromosome organization of the protozoan Trypanosoma brucei.

The genome of the protozoan Trypanosoma brucei is known to be diploid. Karyotype analysis has, however, failed to identify homologous chromosomes. Having refined the technique for separating trypanosome chromosomes (L. H. T. Van der Ploeg, C. L. Smith, R. I. Polvere, and K. Gottesdiener, Nucleic Acids Res. 17:3217-3227, 1989), we can now provide evidence for the presence of homologous chromosomes. By determining the chromosomal location of different genetic markers, most of the chromosomes (14, excluding the minichromosomes), could be organized into seven chromosome pairs. In most instances, the putative homologs of a pair differed in size by about 20%. Restriction enzyme analysis of chromosome-sized DNA showed that these chromosome pairs contained large stretches of homologous DNA sequences. From these data, we infer that the chromosome pairs represent homologs. The identification of homologous chromosomes gives valuable insight into the organization of the trypanosome genome, will facilitate the genetic analysis of T. brucei, and suggests the presence of haploid gametes.