Restriction Fragments of Human Chromosome 21 (Pulsed-Field Gel Electrophoreis/Physcal Mapplng/Genomlc Anon) JESUS SAINZ*, LARYSA Pevnyt, YUE Wul, CHARLES R

Restriction Fragments of Human Chromosome 21 (Pulsed-Field Gel Electrophoreis/Physcal Mapplng/Genomlc Anon) JESUS SAINZ*, LARYSA Pevnyt, YUE Wul, CHARLES R

Proc. Natl. Acad. Sci. USA Vol. 89, pp. 1080-1084, February 1992 Genetics Distribution of interspersed repeats (Mu and Kpn) on Not I restriction fragments of human chromosome 21 (pulsed-field gel electrophoreis/physcal mapplng/genomlc anon) JESUS SAINZ*, LARYSA PEVNYt, YUE WUl, CHARLES R. CANTOR*t, AND CASSANDRA L. SMITH*1§ *Division of Chemical Biodynamics, Lawrence Berkeley Laboratory and *Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720; and tDepartment of Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, NY 10032 Contributed by Charles R. Cantor, November 7, 1991 ABSTRACT Interspersed repeated sequences (Ala and common 3' end. The number of copies is 50,000-100,000 for Kpn) were used as probes to detect a set of Not I restriction the 3' end and 4000-20,000 for the 5' end (22, 25), with an fragments of human chromosome 21 from the hybrid cell line average distance between elements of30-60 and 150-750 kb, WAV17. Forty different Not I fgments, raging in size from respectively. It is not clear whether the LINE sequences <0.05 megabase (Mb) to 7.0 Mb, were Ientied. The total containing and lacking the 5'-end regions are distributed length ofthese fragments was 47.3 Mb. This length provides an randomly (17). estimate of the minimum size of the chromosome and a From estimates of interspersed repeat copy numbers we minimum number of frments to be ordered to create a can calculate that, on average, each megabase of human complete restriction map. The average length Not I fIrent s DNA should contain 100-300 copies of Alu, 17-32 copies of 1.2 Mb. Ala and Kpn fIgments are not always it: a the 3'-end Kpn sequence, and 1.3-6.7 copies of the 5'-end 2.9-Mb fragment is dee with Kpn but not with Alu, and 13 Kpn sequence. Thus, the use ofAlu repeats as hybridization agments, ranging from <0.05 Mb to 5.6 Mb, are detected probes should detect, with high probability, all the chromo- with Alm but not with Kpn; the 26 reaing frgments, some 21 Not I restriction fragments. For instance, Poisson covering 75% (35.3 Mb) of the total length, are d tdwh statistics predict that the probability of not finding an Alu both repetitive probes. The presence ofso many n cnt sequence on a 1.0-Mb fragment is <4 x 10-". The proba- fragments and the high variation of the hybrtion gnal bility of not finding a 3'-end Kpn sequence on a 1.0-Mb intensities of the frgents suggest a very nonuniform distri- fragment is <6 x 10-8, and the probability of not finding a bution of Kpn and Alu repeats. 5'-end Kpn sequence will be somewhere in the range of 0.001-0.26. Thus, when used in hybridization these probes Human chromosome 21, the smallest human chromosome, is should, in concert, detect virtually all the Not I fragments of an appropriate model for large-scale chromosome mapping. human chromosome 21, unless there are peculiar regions This acrocentric chromosome contains rRNA-encoding where some repeats are not tolerated. DNA repeats and contains genes responsible for Down syndrome (1-3), some forms of familial Alzheimer disease (4-6), and amyotrophic lateral sclerosis (7). MATERIALS AND METHODS Previous restriction mapping efforts on smaller chromo- Hybrid cell line WAV17 is a mouse cell line that contains somes were aided considerably by the a priori knowledge of human chromosome 21 as its only human component. all the DNA fragments that needed to be ordered. The WAV17 has an average of three copies of human chromo- restriction enzyme Not I cleaves the human genome into some 21 per cell (ref. 26; data not shown). DNA from this cell fragments with an estimated average size of 1.0 megabase line was purified in agarose blocks (inserts), digested with the (Mb) (8, 9). Since human chromosome 21 is estimated to be restriction enzyme Not I (10 units ofenzyme per ,.g ofDNA), %"51 Mb (10, 11), this chromosome should contain roughly 51 and fractionated by PFGE (27, 28). Size standards were Not I fragments. Chromosome 21 Not I restriction fragments, bacteriophage A DNA concatemers, and Saccharomyces fractionated by pulsed-field gel electrophoresis (PFGE; refs. cerevisiae, Pichia, and Schizosaccharomyces pombe chro- 12 and 13), can be identified in human-rodent hybrid cell line mosomal DNAs (refs. 29 and 30; T.-Y. Zhang, S. Ringquist, DNA by using human-specific interspersed repeated se- J.-B. Fan, and C.L.S., unpublished data). Southern blotting quences as hybridization probes (9). and hybridization conditions were as described (27, 28). Two Short and long interspersed repeated DNA sequences different Alu probes were used. Plasmid pX-H contains a (SINEs and LINEs, respectively) are classified by their 2.0-kb Xba I/HindIII fragment that has five different Alu length (14, 15). Some studies suggest that there is a comple- Plasmid Blur8 has 300 bp of DNA containing mentary distribution of the SINEs and LINEs in the human sequences (31). SINE is the Alu a single Alu sequence (32). The Alu sequences were analyzed genome (16-18). In human DNA, the major by using an Alu identification program.1 This program com- sequence family (19). The human genome contains 300,000 to pares Alu sequences with an Alu consensus sequence and six 900,000 copies of the 300-base-pair (bp) Alu sequence (20- different Alu subfamilies (34). Three Kpn probes were used 22). The Alu family represents 3-9% of the haploid genome 1 ref. kb) contains 1.2 kb of (21). The average distance between Alu sequences is 3-10 (see Fig. and 35). Tey--C (1.6 distributed 3'-end Li sequence, Tey-D (1.5 kb) contains 0.8 kb of kilobases (kb), and copies appear to be widely sequence adjacent to TeyC, and ALTO (0.45 kb) contains across the entire human genome (14, 23, 24). near the 5' end ofan Li element including features The major human LINE family is the 6.4-kb Li or Kpn sequences sequence. Many Kpn repeats have deletions and rearrange- at the 5' end, but most of them share a Abbreviations: Mb, megabase(s); PFGE, pulsed-field gel electro- ments, especially phoresis; LINE, long interspersed repeated DNA sequence; SINE, short interspersed repeated DNA sequence. The publication costs ofthis article were defrayed in part by page charge §To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" Milosavjlevic, A., Jurka, J. & Monardo, P. Human Alu Sequence in accordance with 18 U.S.C. §1734 solely to indicate this fact. Identification Via Electronic Mail (Internet: [email protected]). 1080 Downloaded by guest on September 23, 2021 Genetics: Sainz et al. Proc. Natl. Acad. Sci. USA 89 (1992) 1081 TE-i- C Te>./- D the same number of bands as probe Blur8. However, two 'IJLTR bands (0.20 and 0.087 Mb) were detected only with probe 3' end V pX-H (Table 1). The larger number of bands detected with pX-H is consistent with the fact that it contains five different 3' I I -- --- I,-, I.s 'I I 15 Alu sequences (three, one, and one copy of subfamilies J, Sp, and Sc, respectively). The pX-H Alu sequences have 68-84% -2 kb l Li internal 6 kb 8 kb 0.3 kb element homology with the Alu consensus sequence (34). The Alu truncation probes detected a total of39 Not I bands, ranging in size from novel end 0.014 to 7.0 Mb, with a total apparent length of44.4 Mb. The Insertion largest size standard used was the 5.7-Mb chromosome I of FIG. 1. Location of the Kpn probes (TeyC, Tey-D, OILTR) on an Sc. pombe (30); the size of the largest Not I fragment from Li element. Shaded portion indicates an inserted Li element lacking chromosome 21 is >5.7 Mb. Digestion ofthis Not I fragment the 5' end. The figure is a redrawn composite from Rogan et al. (35). with other restriction enzymes revealed its true size of 7 Mb (R. Oliva, J.S., H. Ichikawa, M. Murata, M. Ohki, and that resemble the long terminal repeat of transposable DNA C.L.S., unpublished data). The average size of the bands elements. Probes were labeled with [32P]dCTP by random detected with pX-H is 1.1 Mb. Seventy-two percent of the priming (36). Band signal intensities were quantitated directly bands detected with the Alu probe pX-H are <1 Mb. from the labeled membrane by using a Molecular Dynamics As expected, the Kpn probes detected a smaller number of 400A Phosphorlmager image scanner. Differences in signal bands than the Alu probes. All the bands detected using the intensity for the same probe hybridized to different mem- Kpn probes were coincident in size with the bands detected branes are seen due to experimental factors such as the using Alu probes, with one exception-a 2.9-Mb band (Table amount ofDNA loaded, time ofexposure, quality ofthe blot, 1). The 3'-end Kpn probe Try-C detected 27 bands ranging in and specific activity of the probe. These differences were size from 0.14 to 7.0 Mb with a total apparent length of 38.2 compensated for by integrating the total amount of signal on Mb and an average size of 1.4 Mb (Fig. 2B). Almost all ofthe each membrane with a particular probe before calculating the large Not I bands were detected with both Alu and Kpn relative amount of signal assigned to individual bands.

View Full Text

Details

  • File Type
    pdf
  • Upload Time
    -
  • Content Languages
    English
  • Upload User
    Anonymous/Not logged-in
  • File Pages
    5 Page
  • File Size
    -

Download

Channel Download Status
Express Download Enable

Copyright

We respect the copyrights and intellectual property rights of all users. All uploaded documents are either original works of the uploader or authorized works of the rightful owners.

  • Not to be reproduced or distributed without explicit permission.
  • Not used for commercial purposes outside of approved use cases.
  • Not used to infringe on the rights of the original creators.
  • If you believe any content infringes your copyright, please contact us immediately.

Support

For help with questions, suggestions, or problems, please contact us