Gene, 111 (1992) 131-134 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00
131
GENE 06307
Physical mapping of the Saccharomyces cerevisiae Ap4A phosphorylase I-encoding gene by the Achilles' cleavage method (Recombinant DNA; diadenosine 5', 5 ' " - P m,p4-tetraphosphate; chromosome III; APA1 gene; yeast; chromosome fragmentation)
Preston N. GarrisonI, Michael K o o b b and LarryD. BarnesI o Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78284-7760 (U.S.A.), and b McArdle Laboratory for Cancer Research. University of Wisconsin, Madison, WI 53706 (U.S.A.) TeL (608)262-2047 or 1259; Fax (608)262-2824 Received by G.P, Livi: 25 September 1991 Revised/Accepted: 12 November/13 November 1991 Received at publishers: 2 December 1991
SUMMARY
Lacl-mediated Achilles' cleavage (AC) is a method for selective fragmentation of chromosomes at special lac operator sites introduced by gene targeting methods [Koob and Szybalski, Science 250 (1990) 271-273]. The Saccharomyces cerevisiae APA I gene, coding for diadenosine 5', 5"-P 1 p4 tetraphosphate phosphorylase I, has previously been shown to be located on chromosome III [Kaushal et al., Gene 95 (1990) 79-84]. We have now used the AC method to map APAI gene to a site 44 kb from the left terminus of the chromosome, between the HIS4 and HML genes. This location was confirmed by the comparison of restriction maps of the APA1 gene region to published restriction maps of chromosome ill.
INTRODUCTION
Achilles' (or Achilles' heel) cleavage (AC) is a general method for combining the specificities of restriction enzymes with those of other DNA-binding molecules (Koob et al., 1988). LacI-mediated AC has been shown to be capable of completely cleaving the S. cerevisiae genome specifically at a single symmetric lac operator (lacOs) (Sadler et al., 1983) that had been introduced at the URA3 gene on chromosome V (Koob and Szybalski, 1990). Analysis of the resulting chromosomal fragments precisely mapped the
Correspondence to: Dr. L.D. Barnes, Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78284-7760 (U.S.A.) Tel. (512)567-3730; Fax (521)567-2490. Abbreviations: AC, Achilles' (or Achilles' heel) cleavage (see Koob et al., 1988); Ap, ampicillin; ApAA,diadenosine 5', 5"-P I, P4-tetraphosphate;
AC site, thus demonstrating the potential usefulness of AC as a tool for the physical mapping of genomes. The S. cerevisiaeAPAI gene encodes the major isozyme ofAp4A phosphorylase I, the degradative enzyme for Ap4A and other dinucleoside-5', 5"-tetraphosphates in yeast (Plateau et al., 1989; Kaushal et al., 1990). We have previously referred to APAI as DTP (Kaushal et al., 1990), but we will now use the designation APA 1 (Plateau et al., 1989). The APA1 gene was previously mapped to chromosome III by hybridization to a pulsed-field gel of yeast chromosomes (Kaushal et al., 1990). Here, the gene is further localized on chromosome lII by the AC method.
APA 1 gene, yeast gene encoding Ap,tA phosphorylase I; EtdBr, ethidium bromide; HML, yeast silent mating-typegene on the left arm of chromosome ill; kb, kilobase(s) or 1000bp; LacOs, symmetric synthetic lac operator (Sadler et al., 1983 and Koob et al., 1988); PFGE, pulsed-field gel electrophoresis; R resistance/resistant; S., Saccharomyces;Tc, tetracycline; u, unit(s).
132 that the AC site was between the sites of the two probes, since P2 detected the small fragment of chromosome III, and Pl detected the large fragment (Fig. 2 and data not shown, respectively). Hybridization of a probe from the ApR-encoding gene detected only the large fragment of chromosome III, confirming that A P A I is transcribed toward the left telomere (data not shown).
EXPERIMENTALAND DISCUSSION
(a) AC mapping of the APAI gene To create an AC site closely linked to theAPA1 gene, an integrating yeast plasmid was constructed containing lacO~, APAI and the URA3 gene (Fig. 1). This plasmid was cleaved within APAI and used to generate integrative yeast transformant~ of strain YNN295 (Vollrath and Davis, 1987) containing the AC site between the duplicated APA I gene copies. Chromosomes from one of these strains were analyzed by AC followed by separation with PFGE (Fig. 2). The EtdBr-stained gels showed that, as expected, chromosome III was specifically cleaved in only the transformant (Fig. 2, lanes 3-5). The size of the chromosome fragments indicated that APAI is about 44 kb from one end of chromosome Ill, including a correction of 4 kb for the plasmid sequences between the AC site and the APAI gene copy closest to the left telomere. At this point it was possible to determine that APA1 is on the left arm of the chromosome by comparing the known restriction map data on the APA 1 region (Kaushal et ai., 1990, Plateau et al., 1989) to recently published restriction maps of chromosome III (Huberman et al., 1988; Newlon et al., 1986; Yoshikawa and Isono, 1990).
(c) Conclusions (1) The APA1 gene in strain YNN295 is located on the left arm of chromosome III about 44 kb from the chromosome end. The B a m H I site in A P A I is identified as equivalent to the site 36 kb from the left end of the map of Yoshikawa and Isono (1990), and the site between the A6C and B9G B a m H I fragments (Huberman et al., 1988), 23 kb from H M L (Newlon et al., 1986). APA1 is transcribed toward the left telomere, and probably represents transcript 23 of Yoshikawa and Isono (1990). The location and orientation have been confirmed by sequencing the B9G fragment and proximal region of the A6C fragment (R. McKee, C. Lewis, B. Pearson and L. Fuller, personal communication), which gave a sequence overlapping the A6C-B9G junction with only minor differences from the published APAI sequences (Kaushal et al., 1990; Plateau et al., 1989). The distance from APAI to the left terminus measured by AC appears to be slightly larger than the corresponding distance on the map of Yoshikawa and lsono (1990). However, as they noted, their set of clones may not have contained a clone with the chromosome end, since none of their clones near the left end contained a telomere X element. Button and Astell (1986) reported an X element at the left end of chromosome III and a distance of 17 kb from
(b) Application of hybridization probes On the basis of this localization, two hybridization probes expected to be from opposite sides of APAI were prepared from a plasmid clone that was isolated by Newlon et al. (1986) during derivation of one of the chromosome III restriction maps (Huberman et al., 1988; Newlon et al., 1986) (Fig. 1). Hybridization with these probes confirmed EcoRl
HhaI, Hadl
EcoRI
YIplacO.APA1 paI
nl
C h r o m o s o m e Ill BgG (6.5 kb) Left terminus BamHI [ ..,.,,.,_ ~l BamHI EcoRl EcoRI I S l~U j i
........l../....v...J..,J........J.... f f
I"-' kb4
8.12 k b
EcoRl
Hpal
BamHI
X n Hp.al BamHI
I I
: APA1
A6C (6.5 kb) EcoRl
I kb XhoI
I BamHI f
Centromere
f
I*-" 2, kb-4
Fig. 1. The APAl targetingplasmidand the site of integrationon chromosomeIll. YlplacO.APA1 was constructedfromApa1-,4atll fragmentsof YIpSlacO (Koob and Szybalski, 1990; Sambrooket al., 1989)and YEp352-DTP2 (Kaushalet al., 1990). The ~.sequencederivesfrom the left ann of the original ).gtlI clonecontainingAPAI. The locationof APAI on chromosomeIll was deducedas describedin sectionsa and b. PI and P2 are the fragmentsused as probes for the AC analysis.The locationof restriction sites in the A6C and B9G segmentsis based on the data of Newlonet al. (1986), Huberman et al. (1988), and Yoshikawaand lsono (1990), and was confirmedby our data (not shown)and the sequenceof B9G. The distances to HIS4 and HML are based on Newlonet al. (1986) and are also compatiblewith the data of Yoshikawa and Isono (1990). The distanceto the terminusfrom HML is based on the data of Button and Asteil (1986)o
133
i~
(360 kb) III (316 kb) m a
: ~< ~'!:~::
VI
I
144 kb) IIIb
~
1
2
3
4
5
6
1
2
3
4
5
Fig. 2. Lacl-mediated AC fragmentation of chromosome ii! at the APA/-targeted laeO, sequence. Lanes 1-6 on the left are an EtdBr-stained PFGE gel. Yeast strain YNN295 (~ ura3-52 lys2 adel ade2 his7 trpl.A1) was transformed with Hpal-cut YlplacO.APAI using a lithium acetate procedure (Schiestl and Gietz, 1989). One transformant, PGYI 11, was analyzed by Lacl-mediated AC. The agarose microbead-emhedded chromosomes were methylated with 1/fl M'Hhal (24 u, New England Biolabs) either in the presence (lanes 3-5) or absence (lanes 1 and 2) of Lacl (approx. 2.4 btg//Jl) and then digested with 10 u of Haeil. All procedures were as described by Koob and Szybalski (1990). The treated microbeads were then loaded on a 1% agarose gel, and PFGE was performed at 14°C, 150 V, with a 40 s switch-time for 14 h on a CHEF-DR II apparatus (BioRad). DNA is from: lanes: i, fully methylated YNN295, 2, fully methylated PGYI 11; 3, AC-proeessed YNN295; 4,5, AC-processed PGYI 11 using 0.5/~1 and 1/~1 LacI, respectively', 6, concatemer standard (Clontech). The chromosome bands are identified by the Roman numerals on the left. Ilia and Illb are the AC fragments of chromosome III. Lanes 1-$ (right hand panel) represent the hybridization of probe 2 (P2) to a blot of the gel (lanes 1-5, lefthand panel). The DNA was fragmented by acid depurination followed by base cleavage and transferred by capillary blotting onto a Nytran membrane (Schleicher & Schuell), as described by the supplier. The EcoRl fragment labeled P2 in Fig. 1 was purified from a low-melting-agarose gel using Clean-a-Gene (Andes Scientific) and used to prepare a digoxigenin-labeled random-primed probe using a Genius kit (Boehringer, Mannheim). Hybridization, washes, and detection with alkaline phosphatase-coupled antibody and Lumi-Phos 530 (Boehringer) were performed according to the kit instructions with minor modifications.
the BamHI site on the right of HML to the terminus (Fig. 1), which would give a composite APA l-left-terminus distance of 39 kb, in reasonable agreement with the 44-kb distance measured by the AC procedure. (2) The ability of LacI-mediated AC to cleave genomes at a single site can be exploited to quickly determine the chromosomal location and orientation of genes, as we have demonstrated with S. cerevisiae APAI. This approach to physical mapping should be particularly useful for organisms that are amenable to homologous genetic recombination and that have chromosomes that can be readily resolved by PFGE. Fragments of larger chromosomes that have been converted to yeast artificial chromosomes should also be readily analyzable by the AC procedure.
ACKNOWLEDGEMENTS
We thank Carol Newlon for a plasmid and Ray McKee for sharing sequence data before publication. The sequencing of B9G by McKee et al. was part of the project to sequence chromosome III of the Biotechnology Action Programme of the Commission of the European Communities. This work was supported by grant GM-27220 (to L.D.B.) from the National Institute of General Medical Sciences. The AC procedures, as carried out by M.K. in the McArdle Laboratory, University of Wisconsin, were supported by the NIH grant No. 1RO1HG00379-01 from the National
Center for Human Genome Research to Dr. W. Szybalski, who initiated the present collaborative effort.
REFERENCES Button, L.L. and Astell, C.R.: The Saccharomyces cerevisiae chromosome III left telomere has a type X, hut not a type Y', ARS region. Mol. Cell. Biol. 6 (1986) 1352-1356. Huherman, J.A., Zhu, J., Davis, LR. and Newlon, C.S.: Close association of a DNA replication origin and an ARS element on chromosome Ill of the yeast, Saceharomyces cerevisiae. Nucleic Acids Res. 16 (1988) 6373-6384. Kaushal, V., Avila, D.M., Hardies, S.C. and Barnes, L.D.: Sequencing and enhanced expression &the gene encoding diadenosine 5', 5"-P g, P%tetraphosphate (Ap4A) phosphorylase in Saccharomyces cerevisiae. Gene 95 (1990) 79-84. Koob, M., Grimes, E. and Szybalski, W.: Conferring operator specificity on restriction endonucleases. Science 241 (1988) 1084-1086. Koob, M. and Szybalski, W.: Cleaving yeast and Escherichta coilgenomes at a single site. Science 250 (1990) 271-273. Newlon, C.S., Green, R.P., Hardeman, K.J., Kim, K.E., Lipchitz, L.R., Palzkill, T.G., Synn, S. and Woody, S.T.: Structure and organization of yeast chromosome IIL In: Hicks, J. (Ed.), Yeast Cell Biology. Alan Liss, New York, 1986, pp. 211-223. Plateau, P., Fromant, M., Schmitter, J.-M., Buhler, J.-M. and Blanquet, S.: Isolation, characterization, and inactivation of the APAI gene encoding yeast diadenosine 5', 5 '"-PI, p4.tetraphosphat e phosphorylase. J. Bacteriol, 171 (1989) 6437-6445. Sadler, J.R., Sasmor, H. and Betz, J.L.: A perfectly symmetric lac operator binds the lac represser very tightly. Prec. Natl. Acad. Sci. USA 80 (1983) 6785-6789.
134 Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Sehiestl, R.H. and Gietz, R.D.: High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet. 16 (1989) 339-346. Vollrath, D. and Davis, R.W.: Resolution of DNA molecules greater than
5 megabases by contour-clamped homogeneous electric fields. Nucleic Acids Res. 15 (1987) 7865-7876. Yoshikawa, A. and Isono, K.: Chromosome III of Saceharomyces cerevisiae: an ordered clone bank, a detailed restriction map and analysis of transcripts suggest the presence of 160 genes. Yeast 6 (1990) 383-401.
131
GENE 06307
Physical mapping of the Saccharomyces cerevisiae Ap4A phosphorylase I-encoding gene by the Achilles' cleavage method (Recombinant DNA; diadenosine 5', 5 ' " - P m,p4-tetraphosphate; chromosome III; APA1 gene; yeast; chromosome fragmentation)
Preston N. GarrisonI, Michael K o o b b and LarryD. BarnesI o Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78284-7760 (U.S.A.), and b McArdle Laboratory for Cancer Research. University of Wisconsin, Madison, WI 53706 (U.S.A.) TeL (608)262-2047 or 1259; Fax (608)262-2824 Received by G.P, Livi: 25 September 1991 Revised/Accepted: 12 November/13 November 1991 Received at publishers: 2 December 1991
SUMMARY
Lacl-mediated Achilles' cleavage (AC) is a method for selective fragmentation of chromosomes at special lac operator sites introduced by gene targeting methods [Koob and Szybalski, Science 250 (1990) 271-273]. The Saccharomyces cerevisiae APA I gene, coding for diadenosine 5', 5"-P 1 p4 tetraphosphate phosphorylase I, has previously been shown to be located on chromosome III [Kaushal et al., Gene 95 (1990) 79-84]. We have now used the AC method to map APAI gene to a site 44 kb from the left terminus of the chromosome, between the HIS4 and HML genes. This location was confirmed by the comparison of restriction maps of the APA1 gene region to published restriction maps of chromosome ill.
INTRODUCTION
Achilles' (or Achilles' heel) cleavage (AC) is a general method for combining the specificities of restriction enzymes with those of other DNA-binding molecules (Koob et al., 1988). LacI-mediated AC has been shown to be capable of completely cleaving the S. cerevisiae genome specifically at a single symmetric lac operator (lacOs) (Sadler et al., 1983) that had been introduced at the URA3 gene on chromosome V (Koob and Szybalski, 1990). Analysis of the resulting chromosomal fragments precisely mapped the
Correspondence to: Dr. L.D. Barnes, Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78284-7760 (U.S.A.) Tel. (512)567-3730; Fax (521)567-2490. Abbreviations: AC, Achilles' (or Achilles' heel) cleavage (see Koob et al., 1988); Ap, ampicillin; ApAA,diadenosine 5', 5"-P I, P4-tetraphosphate;
AC site, thus demonstrating the potential usefulness of AC as a tool for the physical mapping of genomes. The S. cerevisiaeAPAI gene encodes the major isozyme ofAp4A phosphorylase I, the degradative enzyme for Ap4A and other dinucleoside-5', 5"-tetraphosphates in yeast (Plateau et al., 1989; Kaushal et al., 1990). We have previously referred to APAI as DTP (Kaushal et al., 1990), but we will now use the designation APA 1 (Plateau et al., 1989). The APA1 gene was previously mapped to chromosome III by hybridization to a pulsed-field gel of yeast chromosomes (Kaushal et al., 1990). Here, the gene is further localized on chromosome lII by the AC method.
APA 1 gene, yeast gene encoding Ap,tA phosphorylase I; EtdBr, ethidium bromide; HML, yeast silent mating-typegene on the left arm of chromosome ill; kb, kilobase(s) or 1000bp; LacOs, symmetric synthetic lac operator (Sadler et al., 1983 and Koob et al., 1988); PFGE, pulsed-field gel electrophoresis; R resistance/resistant; S., Saccharomyces;Tc, tetracycline; u, unit(s).
132 that the AC site was between the sites of the two probes, since P2 detected the small fragment of chromosome III, and Pl detected the large fragment (Fig. 2 and data not shown, respectively). Hybridization of a probe from the ApR-encoding gene detected only the large fragment of chromosome III, confirming that A P A I is transcribed toward the left telomere (data not shown).
EXPERIMENTALAND DISCUSSION
(a) AC mapping of the APAI gene To create an AC site closely linked to theAPA1 gene, an integrating yeast plasmid was constructed containing lacO~, APAI and the URA3 gene (Fig. 1). This plasmid was cleaved within APAI and used to generate integrative yeast transformant~ of strain YNN295 (Vollrath and Davis, 1987) containing the AC site between the duplicated APA I gene copies. Chromosomes from one of these strains were analyzed by AC followed by separation with PFGE (Fig. 2). The EtdBr-stained gels showed that, as expected, chromosome III was specifically cleaved in only the transformant (Fig. 2, lanes 3-5). The size of the chromosome fragments indicated that APAI is about 44 kb from one end of chromosome Ill, including a correction of 4 kb for the plasmid sequences between the AC site and the APAI gene copy closest to the left telomere. At this point it was possible to determine that APA1 is on the left arm of the chromosome by comparing the known restriction map data on the APA 1 region (Kaushal et ai., 1990, Plateau et al., 1989) to recently published restriction maps of chromosome III (Huberman et al., 1988; Newlon et al., 1986; Yoshikawa and Isono, 1990).
(c) Conclusions (1) The APA1 gene in strain YNN295 is located on the left arm of chromosome III about 44 kb from the chromosome end. The B a m H I site in A P A I is identified as equivalent to the site 36 kb from the left end of the map of Yoshikawa and Isono (1990), and the site between the A6C and B9G B a m H I fragments (Huberman et al., 1988), 23 kb from H M L (Newlon et al., 1986). APA1 is transcribed toward the left telomere, and probably represents transcript 23 of Yoshikawa and Isono (1990). The location and orientation have been confirmed by sequencing the B9G fragment and proximal region of the A6C fragment (R. McKee, C. Lewis, B. Pearson and L. Fuller, personal communication), which gave a sequence overlapping the A6C-B9G junction with only minor differences from the published APAI sequences (Kaushal et al., 1990; Plateau et al., 1989). The distance from APAI to the left terminus measured by AC appears to be slightly larger than the corresponding distance on the map of Yoshikawa and lsono (1990). However, as they noted, their set of clones may not have contained a clone with the chromosome end, since none of their clones near the left end contained a telomere X element. Button and Astell (1986) reported an X element at the left end of chromosome III and a distance of 17 kb from
(b) Application of hybridization probes On the basis of this localization, two hybridization probes expected to be from opposite sides of APAI were prepared from a plasmid clone that was isolated by Newlon et al. (1986) during derivation of one of the chromosome III restriction maps (Huberman et al., 1988; Newlon et al., 1986) (Fig. 1). Hybridization with these probes confirmed EcoRl
HhaI, Hadl
EcoRI
YIplacO.APA1 paI
nl
C h r o m o s o m e Ill BgG (6.5 kb) Left terminus BamHI [ ..,.,,.,_ ~l BamHI EcoRl EcoRI I S l~U j i
........l../....v...J..,J........J.... f f
I"-' kb4
8.12 k b
EcoRl
Hpal
BamHI
X n Hp.al BamHI
I I
: APA1
A6C (6.5 kb) EcoRl
I kb XhoI
I BamHI f
Centromere
f
I*-" 2, kb-4
Fig. 1. The APAl targetingplasmidand the site of integrationon chromosomeIll. YlplacO.APA1 was constructedfromApa1-,4atll fragmentsof YIpSlacO (Koob and Szybalski, 1990; Sambrooket al., 1989)and YEp352-DTP2 (Kaushalet al., 1990). The ~.sequencederivesfrom the left ann of the original ).gtlI clonecontainingAPAI. The locationof APAI on chromosomeIll was deducedas describedin sectionsa and b. PI and P2 are the fragmentsused as probes for the AC analysis.The locationof restriction sites in the A6C and B9G segmentsis based on the data of Newlonet al. (1986), Huberman et al. (1988), and Yoshikawaand lsono (1990), and was confirmedby our data (not shown)and the sequenceof B9G. The distances to HIS4 and HML are based on Newlonet al. (1986) and are also compatiblewith the data of Yoshikawa and Isono (1990). The distanceto the terminusfrom HML is based on the data of Button and Asteil (1986)o
133
i~
(360 kb) III (316 kb) m a
: ~< ~'!:~::
VI
I
144 kb) IIIb
~
1
2
3
4
5
6
1
2
3
4
5
Fig. 2. Lacl-mediated AC fragmentation of chromosome ii! at the APA/-targeted laeO, sequence. Lanes 1-6 on the left are an EtdBr-stained PFGE gel. Yeast strain YNN295 (~ ura3-52 lys2 adel ade2 his7 trpl.A1) was transformed with Hpal-cut YlplacO.APAI using a lithium acetate procedure (Schiestl and Gietz, 1989). One transformant, PGYI 11, was analyzed by Lacl-mediated AC. The agarose microbead-emhedded chromosomes were methylated with 1/fl M'Hhal (24 u, New England Biolabs) either in the presence (lanes 3-5) or absence (lanes 1 and 2) of Lacl (approx. 2.4 btg//Jl) and then digested with 10 u of Haeil. All procedures were as described by Koob and Szybalski (1990). The treated microbeads were then loaded on a 1% agarose gel, and PFGE was performed at 14°C, 150 V, with a 40 s switch-time for 14 h on a CHEF-DR II apparatus (BioRad). DNA is from: lanes: i, fully methylated YNN295, 2, fully methylated PGYI 11; 3, AC-proeessed YNN295; 4,5, AC-processed PGYI 11 using 0.5/~1 and 1/~1 LacI, respectively', 6, concatemer standard (Clontech). The chromosome bands are identified by the Roman numerals on the left. Ilia and Illb are the AC fragments of chromosome III. Lanes 1-$ (right hand panel) represent the hybridization of probe 2 (P2) to a blot of the gel (lanes 1-5, lefthand panel). The DNA was fragmented by acid depurination followed by base cleavage and transferred by capillary blotting onto a Nytran membrane (Schleicher & Schuell), as described by the supplier. The EcoRl fragment labeled P2 in Fig. 1 was purified from a low-melting-agarose gel using Clean-a-Gene (Andes Scientific) and used to prepare a digoxigenin-labeled random-primed probe using a Genius kit (Boehringer, Mannheim). Hybridization, washes, and detection with alkaline phosphatase-coupled antibody and Lumi-Phos 530 (Boehringer) were performed according to the kit instructions with minor modifications.
the BamHI site on the right of HML to the terminus (Fig. 1), which would give a composite APA l-left-terminus distance of 39 kb, in reasonable agreement with the 44-kb distance measured by the AC procedure. (2) The ability of LacI-mediated AC to cleave genomes at a single site can be exploited to quickly determine the chromosomal location and orientation of genes, as we have demonstrated with S. cerevisiae APAI. This approach to physical mapping should be particularly useful for organisms that are amenable to homologous genetic recombination and that have chromosomes that can be readily resolved by PFGE. Fragments of larger chromosomes that have been converted to yeast artificial chromosomes should also be readily analyzable by the AC procedure.
ACKNOWLEDGEMENTS
We thank Carol Newlon for a plasmid and Ray McKee for sharing sequence data before publication. The sequencing of B9G by McKee et al. was part of the project to sequence chromosome III of the Biotechnology Action Programme of the Commission of the European Communities. This work was supported by grant GM-27220 (to L.D.B.) from the National Institute of General Medical Sciences. The AC procedures, as carried out by M.K. in the McArdle Laboratory, University of Wisconsin, were supported by the NIH grant No. 1RO1HG00379-01 from the National
Center for Human Genome Research to Dr. W. Szybalski, who initiated the present collaborative effort.
REFERENCES Button, L.L. and Astell, C.R.: The Saccharomyces cerevisiae chromosome III left telomere has a type X, hut not a type Y', ARS region. Mol. Cell. Biol. 6 (1986) 1352-1356. Huherman, J.A., Zhu, J., Davis, LR. and Newlon, C.S.: Close association of a DNA replication origin and an ARS element on chromosome Ill of the yeast, Saceharomyces cerevisiae. Nucleic Acids Res. 16 (1988) 6373-6384. Kaushal, V., Avila, D.M., Hardies, S.C. and Barnes, L.D.: Sequencing and enhanced expression &the gene encoding diadenosine 5', 5"-P g, P%tetraphosphate (Ap4A) phosphorylase in Saccharomyces cerevisiae. Gene 95 (1990) 79-84. Koob, M., Grimes, E. and Szybalski, W.: Conferring operator specificity on restriction endonucleases. Science 241 (1988) 1084-1086. Koob, M. and Szybalski, W.: Cleaving yeast and Escherichta coilgenomes at a single site. Science 250 (1990) 271-273. Newlon, C.S., Green, R.P., Hardeman, K.J., Kim, K.E., Lipchitz, L.R., Palzkill, T.G., Synn, S. and Woody, S.T.: Structure and organization of yeast chromosome IIL In: Hicks, J. (Ed.), Yeast Cell Biology. Alan Liss, New York, 1986, pp. 211-223. Plateau, P., Fromant, M., Schmitter, J.-M., Buhler, J.-M. and Blanquet, S.: Isolation, characterization, and inactivation of the APAI gene encoding yeast diadenosine 5', 5 '"-PI, p4.tetraphosphat e phosphorylase. J. Bacteriol, 171 (1989) 6437-6445. Sadler, J.R., Sasmor, H. and Betz, J.L.: A perfectly symmetric lac operator binds the lac represser very tightly. Prec. Natl. Acad. Sci. USA 80 (1983) 6785-6789.
134 Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Sehiestl, R.H. and Gietz, R.D.: High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet. 16 (1989) 339-346. Vollrath, D. and Davis, R.W.: Resolution of DNA molecules greater than
5 megabases by contour-clamped homogeneous electric fields. Nucleic Acids Res. 15 (1987) 7865-7876. Yoshikawa, A. and Isono, K.: Chromosome III of Saceharomyces cerevisiae: an ordered clone bank, a detailed restriction map and analysis of transcripts suggest the presence of 160 genes. Yeast 6 (1990) 383-401.
