Dataset Information

Site-Specific Sequence Exchange Between Homologous and Non-homologous Chromosomes.

ABSTRACT: Transgene integration typically takes place in an easy-to-transform laboratory variety before the transformation event is introgressed through backcrosses to elite cultivars. As new traits are added to existing transgenic lines, site-specific integration can stack new transgenes into a previously created transgenic locus. In planta site-specific integration minimizes the number of segregating loci to assemble into a breeding line, but cannot break genetic linkage between the transgenic locus and nearby undesirable traits. In this study, we describe an additional feature of an in planta gene-stacking scheme, in which the Cre (control of recombination) recombinase not only deletes transgenic DNA no longer needed after transformation but also mediates recombination between homologous or non-homologous chromosomes. Although the target site must first be introgressed through conventional breeding, subsequent transgenes inserted into the same locus would be able to use Cre-mediated translocation to expedite a linkage drag-free introgression to field cultivars.

SUBMITTER: Yin Q

PROVIDER: S-EPMC8850970 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Publications

Site-Specific Sequence Exchange Between Homologous and Non-homologous Chromosomes.

Yin Qian Q Li Ruyu R Ow David W DW

Frontiers in plant science 20220203

Transgene integration typically takes place in an easy-to-transform laboratory variety before the transformation event is introgressed through backcrosses to elite cultivars. As new traits are added to existing transgenic lines, site-specific integration can stack new transgenes into a previously created transgenic locus. <i>In planta</i> site-specific integration minimizes the number of segregating loci to assemble into a breeding line, but cannot break genetic linkage between the transgenic lo ...[more]

PMID: 35185992

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Project description:BackgroundCondensation differences along the lengths of homologous, mitotic metaphase chromosomes are well known. This study reports molecular cytogenetic data showing quantifiable localized differences in condensation between homologs that are related to differences in accessibility (DA) of associated DNA probe targets. Reproducible DA was observed for ~10% of locus-specific, short (1.5-5 kb) single copy DNA probes used in fluorescence in situ hybridization.ResultsFourteen probes (from chromosomes 1, 5, 9, 11, 15, 17, 22) targeting genic and intergenic regions were developed and hybridized to cells from 10 individuals with cytogenetically-distinguishable homologs. Differences in hybridization between homologs were non-random for 8 genomic regions (RGS7, CACNA1B, GABRA5, SNRPN, HERC2, PMP22:IVS3, ADORA2B:IVS1, ACR) and were not unique to known imprinted domains or specific chromosomes. DNA probes within CCNB1, C9orf66, ADORA2B:Promoter-Ex1, PMP22:IVS4-Ex 5, and intergenic region 1p36.3 showed no DA (equivalent accessibility), while OPCML showed unbiased DA. To pinpoint probe locations, we performed 3D-structured illumination microscopy (3D-SIM). This showed that genomic regions with DA had 3.3-fold greater volumetric, integrated probe intensities and broad distributions of probe depths along axial and lateral axes of the 2 homologs, compared to a low copy probe target (NOMO1) with equivalent accessibility. Genomic regions with equivalent accessibility were also enriched for epigenetic marks of open interphase chromatin (DNase I HS, H3K27Ac, H3K4me1) to a greater extent than regions with DA.ConclusionsThis study provides evidence that DA is non-random and reproducible; it is locus specific, but not unique to known imprinted regions or specific chromosomes. Non-random DA was also shown to be heritable within a 2 generation family. DNA probe volume and depth measurements of hybridized metaphase chromosomes further show locus-specific chromatin accessibility differences by super-resolution 3D-SIM. Based on these data and the analysis of interphase epigenetic marks of genomic intervals with DA, we conclude that there are localized differences in compaction of homologs during mitotic metaphase and that these differences may arise during or preceding metaphase chromosome compaction. Our results suggest new directions for locus-specific structural analysis of metaphase chromosomes, motivated by the potential relationship of these findings to underlying epigenetic changes established during interphase.

Project description:Although the pericentromeric regions of chromosomes that are enriched in tandemly repeated satellite DNA represent a significant part of eukaryotic genomes, they remain understudied, which is mainly due to interdisciplinary knowledge gaps. Recent studies suggest their important role in genome regulation, karyotype stability, and evolution. Thus, the idea of satellite DNA as a junk part of the genome has been refuted. The integration of data regarding molecular composition, chromosome behaviour, and the details of the in situ organization of pericentromeric regions is of great interest. The objective of this work was a cytogenetic analysis of the interactions between pericentromeric regions from non-homologous chromosomes in mouse spermatocytes using immuno-FISH. We analysed two events: the associations between centromeric regions of the X chromosome and autosomes and the associations between the centromeric regions of the autosomal bivalents that form chromocenters. We concluded that the X chromosome forms temporary synaptic associations with different autosomes in early meiotic prophase I, which can normally be found until the pachytene-diplotene, without signs of pachytene arrest. These associations are formed between the satellite-DNA-rich centromeric regions of the X chromosome and different autosomes but do not involve the satellite-DNA-poor centromeric region of the Y chromosome. We suggest the hypothetical model of X chromosome competitive replacement from such associations during synaptic correction. We showed that the centromeric region of the X chromosome in association remains free of γH2Ax-dependent chromatin inactivation, while the Y chromosome is completely inactivated. This finding highlights the predominant role of associations between satellite DNA-rich regions of different chromosomes, including the X chromosome. We suppose that X-autosomal transient associations are a manifestation of an additional synaptic disorder checkpoint. These associations are normally corrected before the late diplotene stage. We revealed that the intense spreading conditions that were applied to the spermatocyte I nuclei did not lead to the destruction of stretched chromatin fibers of elongated chromocenters enriched in satellite DNA. The tight associations that we revealed between the pericentromeric regions of different autosomal bivalents and the X chromosome may represent the basis for a mechanism for maintaining the repeats stability in the autosomes and in the X chromosome. The consequences of our findings are discussed.

Dataset Information

Site-Specific Sequence Exchange Between Homologous and Non-homologous Chromosomes.

Publications

Site-Specific Sequence Exchange Between Homologous and Non-homologous Chromosomes.

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