Chromosomes

Example 2

A 30× genome coverage of both Xi19 and Robigus was generated. Bio-informatic mapping of the gDNA reads to the IWGSCWGAV02_2BS_scaffold14096, IWGSCWGAV02_2BS_scaffold11627 and the BAC sequence confirmed large structural differences in the region, which explain the observed lack of recombination. Essentially, there were no gDNA-seq reads from Xi19 that mapped onto the BAC sequence of Renan (FIG. 3). Similarly, none of the gDNA-seq reads from Robigus mapped onto scaffold11627 portion located in the interval. This indicates that the gene content of the corresponding genomic regions in susceptible and resistant plants is very divergent and this was probably caused by a local chromosomal rearrangement. Similar structural rearrangements between the Chinese Spring reference genome sequence and the region containing the Fusarium head blight resistance gene (Fhb1) have recently been published (Schweiger et al, 2016).

Example 1

This example describes the generation of a marker-free B. subtilis strain expressing allulose epimerase. Briefly, in a first step, a B. subtilis strain was transformed with a cassette encoding the BMCGD1 epimerase and including an antibiotic resistance marker. This cassette recombined into the Bacillus chromosome and knocked out 8 kb of DNA, including a large sporulation gene cluster and the lysine biosynthesis gene lysA. In a second step, a second cassette was recombined into the B. subtilis chromosome, restoring the lysA gene and removing DNA encoding the antibiotic resistance. E. coli strain 39 A10 from the Keio collection was used to passage plasmid DNA prior to transformation of B. subtilis. The relevant phenotype is a deficiency in the DNA methylase HsdM in an otherwise wild-type K-12 strain of E. coli.

In detail, a cassette of 5120 bp (SEQ ID NO:1; synthetic DNA from IDT, Coralville, Iowa) was synthesized and cloned into a standard ampicillin resistant pIDT vector. The synthetic piece encoded 700 bp upstream of lysA on the B. subtilis chromosome, the antibiotic marker cat (651 bp), the DNA-binding protein lad (1083 bp), and the allulose epimerase (894 bp), and included 700 bp of homology in dacF. This vector was transformed into E. coli strain 39 A10 (Baba et al., 2006), and plasmid DNA was prepared and transformed into B. subtilis strains 1A751 and 1A976.

Transformants were selected on LB supplemented with chloramphenicol. The replicon for pIDT is functional in E. coli but does not work in Gram positive bacteria such as B. subtilis. The colonies that arose therefore represented an integration event into the chromosome. In strain 1A751, the colony morphology on the plates was used to distinguish between single and double recombination events. The double recombination event would knock out genes required for sporulation, whereas the single recombination would not. After three days on LB plates, colonies capable of sporulation were brown and opaque; sporulation-deficient colonies were more translucent.

B. subtilis strain 1A976 with the allulose epimerase cassette is auxotrophic for histidine and lysine and can achieve very high transformation efficiency upon xylose induction. A 1925 bp synthetic DNA (SEQ ID NO:2) was amplified by primers (SEQ ID NO:3, SEQ ID NO:4) and Taq polymerase (Promega). This PCR product encoded the lysA gene that was deleted by the dropping in the epimerase cassette and 500 bp of homology to lad. A successful double recombination event of this DNA should result in colonies that are prototrophic for lysine and sensitive to chloramphenicol; i.e., the entire cat gene should be lost.

Transformants were selected on Davis minimal media supplemented with histidine. Colonies that arose were characterized by PCR and streaking onto LB with and without chloramphenicol. Strains that amplified the introduced DNA and that were chloramphenicol sensitive were further characterized, and their chromosomal DNA was extracted.

Strain 1A751 containing the chloramphenicol resistant allulose was transformed with this chromosomal DNA and selected on Davis minimal media supplemented with histidine. Transformants were streaked onto LB with and without chloramphenicol and characterized enzymatically as described below.

Example 3

We generated and analyzed a collection of 14 early-passage (passage ≤9) human pES cell lines for the persistence of haploid cells. All cell lines originated from activated oocytes displaying second polar body extrusion and a single pronucleus. We initially utilized chromosome counting by metaphase spreading and G-banding as a method for unambiguous and quantitative discovery of rare haploid nuclei. Among ten individual pES cell lines, a low proportion of haploid metaphases was found exclusively in a single cell line, pES10 (1.3%, Table 1B). We also used viable FACS with Hoechst 33342 staining, aiming to isolate cells with a DNA content corresponding to less than two chromosomal copies (2c) from four additional lines, leading to the successful enrichment of haploid cells from a second cell line, pES12 (Table 2).

Two individual haploid-enriched ES cell lines were established from both pES10 and pES12 (hereafter referred to as h-pES10 and h-pES12) within five to six rounds of 1c-cell FACS enrichment and expansion (FIG. 1C (pES10), FIG. 5A (pES12)). These cell lines were grown in standard culture conditions for over 30 passages while including cells with a normal haploid karyotype (FIG. 1D, FIG. 5B). However, since diploidization occurred at a rate of 3-9% of the cells per day (FIG. 1E), cell sorting at every three to four passages was required for maintenance and analysis of haploid cells. Further, visualization of ploidy in adherent conditions was enabled by DNA fluorescence in situ hybridization (FISH) (FIG. 1F, FIG. 5c) and quantification of centromere protein foci (FIG. 1G, FIG. 5D; FIG. 6). In addition to their intact karyotype, haploid ES cells did not harbor significant copy number variations (CNVs) relative to their unsorted diploid counterparts (FIG. 5E). Importantly, we did not observe common duplications of specific regions in the two cell lines that would result in pseudo-diploidy. Therefore, genome integrity was preserved throughout haploid-cell isolation and maintenance. As expected, single nucleotide polymorphism (SNP) array analysis demonstrated complete homozygosity of diploid pES10 and pES12 cells across all chromosomes.

Both h-pES10 and h-pES12 exhibited classical human pluripotent stem cell features, including typical colony morphology and alkaline phosphatase activity (FIG. 2A, FIG. 2B). Single haploid ES cells expressed various hallmark pluripotency markers (NANOG, OCT4, SOX2, SSEA4 and TRA1-60), as confirmed in essentially pure haploid cultures by centromere foci quantification (>95% haploids) (FIG. 2C, FIG. 7). Notably, selective flow cytometry enabled to validate the expression of two human ES-cell-specific cell surface markers (TRA-1-60 and CLDN6¹⁸) in single haploid cells (FIG. 2D). Moreover, sorted haploid and diploid ES cells showed highly similar transcriptional and epigenetic signatures of pluripotency genes (FIG. 2E, FIG. 2F). Since the haploid ES cells were derived as parthenotes, they featured distinct transcriptional and epigenetic profiles of maternal imprinting, owing to the absence of paternally-inherited alleles (FIG. 8).

Haploid cells are valuable for loss-of-function genetic screening because phenotypically-selectable mutants can be identified upon disruption of a single allele. To demonstrate the applicability of this principle in haploid human ES cells, we generated a genome-wide mutant library using a piggyBac transposon gene trap system that targets transcriptionally active loci (FIG. 2G, FIG. 8E), and screened for resistance to the purine analog 6-thioguanine (6-TG). Out of six isolated and analyzed 6-TG-resistant colonies, three harbored a gene trap insertion localizing to the nucleoside diphosphate linked moiety X-type motif 5 (NUDT5) autosomal gene (FIG. 2H). NUDT5 disruption was recently confirmed to confer 6-TG resistance in human cells,⁵¹ by acting upstream to the production of 5-phospho-D-ribose-1-pyrophosphate (PRPP), which serves as a phosphoribosyl donor in the hypoxanthine phosphoribosyltransferase 1 (HPRT1)-mediated conversion of 6-TG to thioguanosine monophosphate (TGMP) (FIG. 2I). Detection of a loss-of-function phenotype due to an autosomal mutation validates that genetic screening is feasible in haploid human ES cells.