Engineered Minichromosomes in Plants

Engineered minichromosomes provide the ability to target transgenes to a defined insertion position for predictable expression on an independent chromosome. This technology promises to provide a means to add many genes to a synthetic chromosome in sequential manner. An additional advantage is that the multiple transgenes will not be inserted into the normal chromosomes and thus will not exhibit linkage drag when converging the transgenes to different germplasm nor will they be mutagenic. Telomere truncation coupled with the introduction of site-specific recombination cassettes has proven to be an easy method to produce minichromosomes. Telomere truncation results from the transformation of plasmids carrying a block of telomere repeats at one end. Minichromosomes consisting of little more than a centromere have been produced for B chromosomes of maize. Such small chromosomes have been studied for their meiotic behavior, which differs from normal sized chromosomes in that homologue pairing is rare or nonexistent and sister chromatid cohesion fails at meiosis I. Potential modifications of the minichromosomes that can address these issues are discussed. Minichromosomes can be recovered from transformed plants that are polyploid or that carry an additional chromosome as the preferred target for truncation. Site-specific recombination has been demonstrated to operate on these terminally located sites. By introducing normal B chromosomes into lines with engineered mini-B chromosomes, the latter can be increased in copy number, which provides the potential to augment the expression of the introduced genes. Because the vast majority of plant species have the same telomere sequence, the truncating transgenes should be effective in most plants to generate engineered minichromosomes. Such chromosomes establish the means to add or subtract multiple transgenes, multigene complexes, or whole biochemical pathways to plants to change their properties for agronomic applications or to use plants as factories for the production of foreign proteins or metabolites.     

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.     

Plant engineered minichromosomes and artificial chromosome platforms

The introduction of telomere sequences during transformation of maize will cause chromosomal truncation. This technique has been used to create minichromosomes. With the simultaneous introduction of site specific recombination cassettes, the ability to add additional genes to the newly formed engineered minichromosome becomes possible. Targeting the supernumerary B chromosome produces truncations at a very high rate and produces minichromosomes with the additional property that they can be dosage manipulated by the reintroduction of full length B chromosomes. The uses of engineered minichromosomes are discussed.     

The chromosomal distribution of microsatellite repeats in the genome of the wolf fish Hoplias malabaricus, focusing on the sex chromosomes

Distribution of 12 mono-, di- and tri-nucleotide microsatellites on the chromosomes of 2 karyomorphs with 2 distinct sex chromosome systems (a simple XX/XY - karyomorph B and a multiple X(1)X(1)X(2)X(2)/X(1)X(2)Y - karyomorph D) in Hoplias malabaricus, commonly referred to as wolf fish, was studied using their physical mapping with fluorescence in situ hybridization (FISH). The distribution patterns of different microsatellites along the chromosomes varied considerably. Strong hybridization signals were observed at subtelomeric and heterochromatic regions of several autosomes, with a different accumulation on the sex chromosomes. A massive accumulation was found in the heterochromatic region of the X chromosome of karyomorph B, whereas microsatellites were gathered at centromeres of both X chromosomes as well as in corresponding regions of the neo-Y chromosome in karyomorph D. Our findings are likely in agreement with models that predict the accumulation of repetitive DNA sequences in regions with very low recombination. This process is however in contrast with what was observed in multiple systems, where such a reduction might be facilitated by the chromosomal rearrangements that are directly associated with the origin of these systems.     

XX/XY System of Sex Determination in the Geophilomorph Centipede Strigamia maritima

We show that the geophilomorph centipede Strigamia maritima possesses an XX/XY system of sex chromosomes, with males being the heterogametic sex. This is, to our knowledge, the first report of sex chromosomes in any geophilomorph centipede. Using the recently assembled Strigamia genome sequence, we identified a set of scaffolds differentially represented in male and female DNA sequence. Using quantitative real-time PCR, we confirmed that three candidate X chromosome-derived scaffolds are present at approximately twice the copy number in females as in males. Furthermore, we confirmed that six candidate Y chromosome-derived scaffolds contain male-specific sequences. Finally, using this molecular information, we designed an X chromosome-specific DNA probe and performed fluorescent in situ hybridization against mitotic and meiotic chromosome spreads to identify the Strigamia XY sex-chromosome pair cytologically. We found that the X and Y chromosomes are recognizably different in size during the early pachytene stage of meiosis, and exhibit incomplete and delayed pairing.     

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.     

Localized Aurora B activity spatially controls non-kinetochore microtubules during spindle assembly

Efficient spindle assembly involves the generation of spatial cues around chromosomes that locally stabilize microtubule (MT) plus-ends. In addition to the small GTPase Ran, there is evidence that Aurora B kinase might also generate a spatial cue around chromosomes but direct proof for this is still lacking. Here, we find that the Aurora B substrate MCAK localizes to MT plus-ends throughout the mitotic spindle, but its accumulation is strongly reduced on MT plus-ends near chromatin, suggesting that a signal emanating from chromosomes negatively regulates MCAK plus-end binding. Indeed, we show that Aurora B is the kinase responsible for producing this chromosome-derived signal. These results are the first to visualize spatially restricted Aurora B kinase activity around chromosomes on an endogenous substrate and explain how Aurora B could spatially control the dynamics of non-kinetochore MTs during spindle assembly.     

Interaction of B chromosomes with A or B chromosomes in segregation in insects

Additional or B chromosomes not belonging to the regular karyotype of a species are found in many animal and plant groups. They form a highly heterogeneous group with respect to their morphology and behaviour both in mitosis and meiosis. Achiasmatic mechanisms that ensure the segregation of a B chromosome from another B chromosome or from an A chromosome are reviewed. An achiasmatic mechanism characterized by the "distance pairing" of segregating univalents at metaphase I was found to be responsible for the preferential segregation of B chromosome univalents in Hemerobius marginatus L. (Neuroptera), and a mechanism characterized by the "touch and go pairing" of segregating univalents was responsible for the highly regular segregation of a B chromosome and the X chromosome in Rhinocola aceris (L.) (Psylloidea, Homoptera). The latter mechanism resulted in the integration of a B chromosome to the A chromosome set as a Y chromosome in a psyllid species Cacopsylla peregrina (Frst.). Furthermore, B chromosomes can disturb the regular segregation of the achiasmatic X and Y chromosomes resulting in the formation of X0/XY polymorphism in a population, which might precede the loss of the Y chromosome. The absence of observations on accurately functioning achiasmatic segregation mechanisms in grasshoppers (Orthoptera) was attributed to the X and B chromosomes, which re-orient one or several times during metaphase I. Apparently, these re-orientations mask any achiasmatic segregation mechanism that might operate during meiotic prophase in these insects.     

Overexpression of the dnaA gene in Escherichia coli B/r: chromosome and minichromosome replication in the presence of rifampin.

The replication of chromosomes and minichromosomes in Escherichia coli B/r was examined under conditions in which the dnaA gene product was overproduced. Increased levels of the DnaA protein were achieved by thermoinduction of the dnaA gene, under the control of the lambda pL promoter, or by cellular maintenance of multicopy plasmids carrying the dnaA gene under the control of its own promoters. Previous work has shown that overproduction of DnaA protein stimulates replication of the chromosomal origin, oriC, but that the newly initiated forks do not progress along the length of the chromosome (T. Atlung, K. V. Rasmussen, E. Clausen, and F. G. Hansen, p. 282-297, in M. Schaechter, F. C. Neidhardt, J. L. Ingraham, and N. O. Kjeldgaard, ed., The Molecular Biology of Bacterial Growth, 1985). In the present study, it was found that overproduction of DnaA protein caused both a two- to threefold increase in the amount of residual chromosome replication and an extended synthesis of minichromosome DNA in the presence of rifampin. The amount of residual chromosome replication was consistent with the appearance of functional replication forks on the majority of the chromosomes. Since the rate of DNA accumulation and the cellular DNA/mass ratios were not increased significantly by overexpression of the dnaA gene, we concluded that the addition of rifampin either enabled stalled replication forks to proceed beyond oriC or enabled new forks to initiate on both chromosomes and minichromosomes, or both.     

Morphology and Meiotic/Mitotic Behavior of B Cromosomes in a Japanese Harvestman, Metagagrella tenuipes (Arachnida: Opiliones): No Evidence for B Accumulation Mechanisms

Earlier, it has been demonstrated that wild populations of a Japanese harvestman Metagagrella tenuipes (Arachnida: Opiliones) are polymorphic for B chromosomes. In this paper, we present results of a study of the morphology and mitotic and meiotic behavior of the Bs. The B chromosomes varied considerably in size and proportion of eu- and heterochromatin. The single nucleolus organizing region, found in males, was located on a chromosome of the A complement. Some intercell variation in number of Bs may be explained by accidental chromosome losses during chromosome preparation. We also found no intertissue variation in number of Bs. There were also no differences in mean number of B chromosomes per individual among males and females, adult and subadult harvestmen. Segregation of Bs in mitotic and meiotic divisions was nonrandom; B chromosomes tended to segregate equally between daughter cells. The results obtained provide no support for the hypothesis of existence of B accumulation mechanism in this species.     

Large deletions result from breakage and healing of P. falciparum chromosomes

The human malaria parasite P. falciparum exhibits extensive strain-dependent chromosomal polymorphisms that have been implicated in the generation of antigenic variability in this organism. These polymorphisms can result in large deletions in chromosomes as determined by pulsed-field gradient gel electrophoresis. We have investigated the molecular basis for extensive deletions in chromosomes 2 and 8 in multiple geographic isolates of this parasite that result in the loss of expression of well-characterized parasite antigens. The structure of these polymorphic chromosomes reveal that a mechanism of chromosome breakage and healing by the addition of telomeric repeats most plausibly accounts for these karyotypes. Furthermore, the orientation of these gene fragments on their truncated chromosomes reveal that the healed chromosome originally associated with centromeric elements is mitotically stable and maintained. A model for the possible role of this mechanism in the complex parasite life-cycle is discussed.     

Molecular-cytogenetic analysis reveals sequence differences between the sex chromosomes of Oreochromis niloticus: evidence for an early stage of sex-chromosome differentiation

Sex determination in the Nile tilapia, Oreochromis niloticus, is primarily genetic, with XX females and XY males. A candidate sex-determining region in the terminal region of the largest chromosome pair has been identified by analysis of meiotic chromosomes. This region shows an inhibition of pairing and synapsis in the XY genotype, but not in XX or YY genotypes, suggesting that recombination is inhibited. Here we show that chromosome microdissection and subsequent amplification by degenerate oligonucleotide-primed PCR (DOP-PCR) can be used to produce in situ hybridization probes to this largest pair of O. niloticus chromosomes. Furthermore, analysis of the comparative hybridization of X and Y chromosome-derived probes to different genotypes provides the first demonstration that sequence differences exist between the sex chromosomes of O. niloticus. This provides further support for the theory that this chromosome pair is related to sex determination and further suggests that the sex chromosomes are at a very early stage of divergence.     

Centromere function and nondisjunction are independent components of the maize B chromosome accumulation mechanism

Supernumerary or B chromosomes are selfish entities that maintain themselves in populations by accumulation mechanisms. The accumulation mechanism of the B chromosome of maize (Zea mays) involves nondisjunction at the second pollen mitosis, placing two copies of the B chromosome into one of the two sperm. The B chromosome long arm must be present in the same nucleus for the centromere to undergo nondisjunction. A centromere, containing all of the normal DNA elements, translocated from the B chromosome to the short arm of chromosome 9 was recently found to be epigenetically silenced for centromeric function. When intact B chromosomes were added to this genotype, thus supplying the long arm, the inactive centromere regained the property of nondisjunction causing the translocation chromosome 9 to be differentially distributed to the two sperm or resulted in chromosome breaks in 9S, occasionally producing new translocations. Translocation of the inactive B centromere to chromosome 7 transferred the nondisjunction property to this chromosome. The results provide insight into the molecular and evolutionary basis of this B chromosome accumulation mechanism by demonstrating that nondisjunction is caused by a process that does not depend on normal centromere function but that the region of the chromosome required for nondisjunction resides in the centromeric region.     

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.     

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.     

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.     

High intrachromosomal similarity of retrotransposon long terminal repeats: evidence for homogenization by gene conversion on plant sex chromosomes?

Retrotransposons are ubiquitous in the plant genomes and are responsible for their plasticity. Recently, we described a novel family of gypsy-like retrotransposons, named Retand, in the dioecious plant Silene latifolia possessing evolutionary young sex chromosomes of the mammalian type (XY). Here we have analyzed long terminal repeats (LTRs) of Retand that were amplified from laser microdissected X and Y sex chromosomes and autosomes of S. latifolia. A majority of X and Y-derived LTRs formed a few separate clades in phylogenetic analysis reflecting their high intrachromosomal similarity. Moreover, the LTRs localized on the Y chromosome were less divergent than the X chromosome-derived or autosomal LTRs. These data can be explained by a homogenization process, such as gene conversion, working more intensively on the Y chromosome.