The haploid liverwort has heteromorphic sex chromosomes, an X chromosome in

The haploid liverwort has heteromorphic sex chromosomes, an X chromosome in the female and a Y chromosome in the male. plants. The chromosome-specific repeat sequences possibly contribute to determining the identity of the Y chromosome in as well as to maintaining genes required for male functions, as in mammals such as human. the presence of the Y1 and Y2 chromosomes has no influence on triggering male development, which is, like in (3C7), (8), and (9C11), that suggest a repetitive structure of sex chromosomes in plants. In interspersed and localized repeat sequences were identified on the sex chromosomes by microdissection and degenerate oligonucleotide primed PCR (DOP-PCR; refs. 4, 5, and 7). Some of these sequences are localized at subtelomeric regions, which are near the pseudoautosomal regions of the X and Y chromosomes (5, 7). However, all of these sequences are found on both the sex chromosomes and also on the autosomes, as yet not indicating any exclusive feature for the respective sex chromosomes. Only in were repeat sequences unique to the Y chromosomes reported, but they are limited to just a few hundred bases (10). Several genes have been isolated from the sex chromosomes of is similar to Aspartame IC50 (E lines; ref. 15) were cultivated on M51C medium (16) at 24C under continuous light. Chromosome Preparation and Fluorescence Hybridization (FISH). Chromosomal preparation and treatments before hybridization were performed as described (15). For FISH, 20 l of a hybridization mixture containing 30 ng biotin-labeled pMM4G7 (15), 1 g salmon testis DNA (Sigma-Aldrich), and 15% formamide in 2 SSC were applied to each slide. The mixture was denatured at 85C for 10 min and transferred to preparations. The preparations were heated at 85C for 10 min on a thermal controller with a slide griddle (PTC-100, MJ Research, Cambridge, MA) and hybridized in a humid chamber at 24C overnight. The signals were detected with avidin-conjugated fluorescein Aspartame IC50 (Roche Diagnostics) and amplified once with biotinylated anti-avidin D and fluorescein avidin DCS (Vector Laboratories). Chromosomes were stained with 1.0 g/ml 4,6-diamidino-2-phenylindole Rabbit polyclonal to HHIPL2 (DAPI) in Vectashield (Vector Laboratories) and observed under a Zeiss Axioplan2 with 01 and 17 filter sets for DAPI and fluorescein, respectively. Fluorescent images were documented by a cooled charge-coupled device (CCD) camera, PentaMax (Princeton Instruments, Trenton, NJ) and analyzed with iplab (Scanalytics, Fairfax, VA) and Adobe Photoshop (Adobe Systems, Mountain View, CA). Restriction Mapping. For mapping with or 2.5C5 ng of PAC plasmid DNAs were hybridized with 32P-labeled probes. The same 2.4-kb (E lines; ref. 15) were used as templates. Primers 5-CAAGAGACGACTGACTCGACTG-3 and 5-TCTCCATCCACGCATTGAAGAG-3 were used for the 2 2.2-kb male genomic library maps to the Y chromosome and that closely related sequences have Aspartame IC50 accumulated in the Y chromosome (15). To map pMM4G7 more precisely, FISH analysis with prometaphase chromosomes was performed (Fig. ?(Fig.1).1). The observed signal was concentrated in only one half of the Y chromosome, whereas the other half remains largely free. This observation suggests that the sequences cloned in pMM4G7 are located in one part of the Y chromosome only. The intensity of the signal and the area of the chromosome covered indicate that these sequences are heavily amplified in the Y chromosome. Figure 1 FISH analysis of a chromosome preparation from male and suggest further variants of these repeat sequences in the Y chromosome, albeit mostly at lower copy numbers and/or a lower degree of similarity. Figure 3 Genomic distribution of repeat sequences related to pMM4G7. (accounts for 1.3C3.3 million base pairs (Mb). Consequently, these Y chromosome-specific repeat sequences contribute approximately one quarter of the 10-Mb long Y chromosome (15). For another independent estimation of the copy number of these repeat sequences in the Y chromosome, we analyzed their representation in the library of male PAC clones. This library, with 33,000 clones and an average insert size of 90 kb, covers the total 280-Mb male genome of 10 times (15). The number of the PAC clones identified with the 2 2.4-kb and AtUn is an ORF of unknown function in (15) made it possible to isolate and analyze such long regions of plant sex chromosomes. The 35-kb-long fragment cloned in pMM4G7 is mostly.

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