Han-Chang Chi, Judy M. Buckingham, A. Christine Munk, Elizabeth Saunders, Rebecca Lobb, Jingmei Liu, Quincey Simmons, Michael R. Altherr, Darrell Ricke, Jung-Rung Wu, and Robert K. Moyzis.
Center for Human Genome Studies and Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545.
Most human telomeric genes are hundreds of kilobases from the chromosome termini, which consist of tandem arrays of the simple repeat sequence (TTAGGG)n.[1] Scrambled arrays of more complex repetitive DNA are located internal to the simple sequence repeat.[2] The telomeric end of chromosome 7q appears, however, to lack significant blocks of subtelomeric repetitive DNA.[3] In order to determine what human genes, if any, are near the 7q terminus, as well as whether or not those genes are affected by the telomeric DNA shortening demonstrated to occur in somatic cells, complete DNA sequencing of a 240 kb YAC, representing the end of this chromosome was performed.
Cosmid contigs representing the entire 240 kb terminal DNA from a human 7q telomeric yeast artificial chromosome clone (HTY-146) were constructed. From seven overlapping cosmids and a 11 kb DNA fragment amplified by polymerase chain reaction, a Sample Sequencing (SASE) approach that rapidly generates aligned sequences was initated (see Ricke et al., this meeting). "Nucleation points" from the cosmids or DNAs for SASE analysis were obtained by 1) end sequencing individual cosmids or DNAs, 2) subcloning of single restriction fragments and end sequencing, 3) sequencing Sau3AI total digest subclones, or 4) end sequencing partial digest random 3 kb subcloned fragments.
By assembling and aligning these nucleation points, 70% sequence coverage is achieved with 98% clone coverage from a one-pass, 96-sample sequencing of a cosmid. From these initial sequences of the SASE clones, oligonucleotides were synthesized and further sequence data obtained by sequencing and primer walking directly off the original cosmid DNA. Both DNA sequence analysis and exon trapping experiments have identified some potentially interesting features dispersed along this terminal region of chromosome.
* This work was funded by the U. S. Department of Energy under contract W-7405ENG-36.
References:
[1] Moyzis, R. K. et al., Proc. Natl. Acad. Sci. USA 85, 6622-6626 (1988).
[2] Riethman, H. C. et al., Proc. Natl. Acad. Sci. USA 86, 6240-6244 (1989).
[3] Riethman, H. C. et al., Genomics 17, 25-32 (1993).