A DNA-library established from microdissected bands 8q23 to 8q24.1 of normal human chromosomes 8 (Lüdecke et al., 1989) was used as a probe for chromosomal in situ suppression (CISS-) hybridization to metaphase chromosomes of man and primates including Hylobates lar and Macaca fuscata. Comparative band mapping as first applied in this study shows the specific visualization of a single subchromosomal region in all three species and thus demonstrates that synteny of the bulk sequences of a specific human chromosome subregion has been conserved for more than 20 million years

A. Jauch

B. Dutrillaux

B. Horsthemke

C. Lengauer

D. Pinkel

H. J. Lüdecke

H.J. Lüdecke

J. Wienberg

J.J. Yunis

M. Klever

M. Small

P. Lichter

P.A. Lalley

R. Stanyon

S.J. O'Brien

T. Cremer

English

Lengauer, Christoph

Lüdecke, H. J.

Wienberg, Johannes

Cremer, Thomas

Horsthemke, B.

Open Access LMU ( Ludwig-Maximilians-Univ. München)

HUMAN EVOLUTION Vol. 6 - N. 1 (67-71) - 1991 C. Lengauer, J. Wienberg & T. Cremer Institut [iir Humangenetik und Anthropologie, Universitdt Heidelberg, Germany H.J. Li~decke &B. Horsthemke Institut fiir Humangenetik, Universitdtsklinikum Essen, Germany Comparative chromosome band mapping in primates by in sito suppression hybridization of band specific DNA microlihraries A DNA-library established from microdissected bands 8q23 to 8q24.1 of normal human chromosomes 8 (Liidecke et al., 1989) was used as a probe for chromosomal in situ suppression (CISS-) hybridi- zation to metaphase chromosomes of man and primates including Hylobates far and Macaca fuscata. Comparative band mapping as first applied in this study shows the specific visualization of a single subchromosomal region in all three species and thus demonstrates that synteny of the bulk sequences of a specific human chromosome subregion has been conserved for more than 20 million years. Introduction Comparisons of chromosome morphology are widely used for phylogenetic recon- structions. Fixation of chromosomal changes are rare events and identical chromosomal rearrangements in different species are considered to reflect a common evolutionary path. Accordingly, ancestral versus derived traits can be established. With few exceptions comparative cytogenetics has indicated a highly conserved chromosome morphology during primate evolution (DuTRmLAUX, 1979; YUNIS & PRAKASH, 1982; STANYON & CHIARELLI, 1983; O'BRIEN et al., 1988). It is widely accepted that chromosome bands whose homology has been suggested on the basis of similar banding patterns reflect synteny of DNA sequences. However, comparative mapping of individual DNA sequences (LALLEY et al., 1990) so far has provided only limited data to unequivocally support his hypothesis. These studies are very time consuming and the mapped sequences only reflect a very small part of a given chromosome band. Recently, we have introduced a new strategy to analyze chromosomal evolution in primates based on chromosomal in situ suppression (CISS-) hybridization (WIENBERC et al., 1990). A series of bacteriophage DNA-libraries established from flow sorted human chromosomes was used to visualize homologies of primate chromosomes directly at the DNA level. However, these chromosome specific DNA libraries did not permit to reconstruct intrachromosomal rearrangements which are supposed to be prominent in the evolution of hominoid karyotypes (DuTRILLAUX, 1979; YUNIS & PRASHAK, 1982; STANYON & CHIARELLI, 1983). An unequivocal demonstration f the homology of individual bands could be based on CISS-hybridization of band specific DNA libraries. Construction of such libraries has become possible by microdissection a d microcloning (LODECKE et al., 1989). Here, we have used a micro-DNA-library established from the distal third of band 8q23 and the proximal two-thirds of sub-band 8q24.1 from normal human chromosomes (LODECKE et al., 1989) for comparative band mapping of human and primate species. For this comparison we have chosen Hylobates lar and Macaca fuscata in the present study. Due to the rapid chromosomal evolution in the lesser apes, it has been exceedingly difficult to establish chromosome homologies both between particular gibbon species and other 9 Edltrice I1 Sedicesimo - Firenze ISSN 0393-9375 68 LENGAUER, WIENBERG, CREMER, LLIDECKE & HORSTHEMKE primates by conventional banding techniques. In contrast, Macaca fuscata shows a much higher degree of chromosomal conservation (STANYON & CHIARELLI, 1983). In particular, CISS-hybridization f a DNA-library from sorted human chromosomes 8 has shown that this chromosome is rearranged in Hylobates lar but has been maintained intact in Macaca fuscata (our unpublished ata). Materials and Methods DNA probes and probe labeling: The microlibrary from chromosome bands 8q23- 8q24.1 was established and characterized byLiidecke et al. (1989). Briefly, 37 dissected chromosome fragments were digested with RsaI and ligated to a SmaI-cut pUC-plasmid. The inserts were than amplified by PCR using the universal M13/pUC sequencing and reverse sequencing primers. In our experiments 100 ng of amplified inserts was again amplified in a 100 ul assay containing 1uM primer each (see above), 10 ~tM Tris-HC1 (pH 8.4), 50 ~tm KC1, 1.5 ~tM MgC12, 0.001% gelatin, 200 0M of dATP, dGTP, dCTP each, 100 0M of dTTP, 150 eM bio-ll-dUTP and 2.5 units of Thermus aquaticus DNA polymerase. After initial denaturation at96~ for 3 rain 25 cycles of PCR were carried out with denaturation at96~ for 1 min, annealing at 47~ for 1 min and extention at 72~ for 2 min. 10 sec. Chromosome preparations: Metaphase chromosome spreads for in situ hybridization experiments were prepared and stored as described by JAUCH et al. (1990). Human chromosomes were derived from phytohemagglutinin (PHA) stimulated peripheral lym- phocytes. Gibbon chromosomes (Hylobates lar) were prepared from a lymphoblastoid cell line kindly provided by T. Ishida (Primate Center, Kyoto, Japan). The karyotype of this cell line was found to be normal diploid (H.lar karyotype: 2n= 44; R. Stanyon, personal communication). Macaca fuscata chromosome spreads from primary kidney cultures (STAN- YON et al., 1990) were kindly provided by R.Stanyon (Istituto di Antropologia, Genoa, Italy). Chromosome banding: Chromosome banding previous to CISS-hybridization was according to Klever et al. (1991). After routine GTG-banding metaphases were photo- graphed (Agfa-Ortho 25), destained with fixative (methanol/acetic acid 3:1) and postfixed with 4% formaldehyde (Merck) in PBS for 15 min. Postfixation was essential to preserve chromosome morphology in the subsequent CISS-hybridization experiments. CISS-hybridization: Sixty ng of PCR labeled probe was used for CISS-hybridization (LIcHTER et al., 1988; PmKEL et al., 1988). Biotin labeled probes were detected with fluoresceine-conjugated vi ine. Signals were amplified once as described by PINKEL et al. (1986) and slides were analysed with a Zeiss photornicroscope III equipped for FITC fluorescence. Photographs were taken on AGFA-CHROME 1000 RS or Kodak T-MAX black and white (400 ASA) films. Results CISS-hybridization of the microlibrary to human lymphocyte metaphase spreads resulted in a complete delineation of band 8q23 and a partial delineation of band 8q24 (Figure la, b). The telomeric band 8q24.3 clearly remained unstained (Figure 2a, b). This Figure 1 - CISS-hybridization f a microlibrary derived from human chromosome bands 8q23-8q24.1 to a human metaphase spread, a) GTG-banding, b) FITC-detection of hybridized microlibrary sequences. Arrows indicate the labeled subregion on chromosome 8 (bands 8q23-24), arrowheads point to a weak hybridization signal on chromosome 9 (band 9q33). Figure 2 - GTG-banding, CISS- hybridization patterns of the mi- crolibrary and idiograms of la- beled chromosomes in man (HSA) (a-c), Hylobates lar (HLA) (d-f) and Macaca ]uscata (MFU) (g-i). The Iabeled regions estab- lished from a series of chromo- somes are indicated in the idio- grams by black dots. 70 LENGAUER, WIENBERG, CREMER, LUDECKE & HORSTHEMKE result is in agreement with the molecular characterization f the microlibrary as previously described (LODECKE et aL, 1989). Unexpectedly, however, an additional faint signal was also evident on chromosome 9q33 (see Discussion). In Hylobates lar and Macaca fuscata a single chromosome subregion was delineated (Figure 2e, h), while site specific signals on other chromosomes could not be detected. The chromosomes with the hybridized subregions were identified on the basis of their G- banding pattern as chromosome 9 in Hylobates lar (Figure 2d) according to the idiogram published by STANYON et aL, (1987), and as chromosome 8 in Macaca [uscata (Figure 2g)according to the numbering system of SMALL et aL (1985). Discussion CISS-hybridization of DNA microlibraries established from microdissected chromo- some regions can be used as a rapid tool to confirm the origin of the bulk DNA sequences from the respective region (Lengauer et al., 1991). This study demonstrates the specific visualization of single chromosome bands using a human microlibrary established after microdissection f bands 8q23-8q24.1. The faint labeling of the additional band 9q33 may be explained by an occassional error in the classification of banded chromosomes u ed for microdissection. Alternatively, cross-hybridization may indicate the presence of conserved sequences in both bands. This latter explanation seems unlikely, since the decoration of complete chromosomes 8 by CISS-hybridization with DNA-libraries established from sorted human chromosomes 8 did not result in any detectable cross-hybridization events on chromosome 9 and vice versa (our unpublished ata). CISS-hybridization of the microlibrary to a single subchromosomal region in Hylo- bates lar and Macaca [uscata, respectively, demonstrates that synteny of the bulk sequences of human chromosome bands 8q23 to 24.1 has been conserved for more than 20 million years. Comparative band mapping as first demonstrated in this study provides new possibilities for cytotaxonomy. As compared to the physical mapping of individual DNA sequences, evolutionary conservation of chromosome bands as well as chromosomal rearrangements with breakpoints in specific bands can be rapidly ascertained. Single copy probes established from such microlibraries may then be used for more detailed studies of possible breakpoints pinpointed within the chromosomal region defined by a microlibrary. Acknowledgements This work was supported in part by the Deutsche Forsc ungsgemeinschaft. We would like to thank R.Stanyon & T.Ishida for providing us with the primate samples, U.Claussen & G.Senger, Erlangen, for collaboration, and A.Wiegenstein for photographic work. C.L. was the recipient of a scholarship from the Konrad-Adenauer-Stiftung and T.C. of a Heisenberg-Stipendium fro  the Deutsche Forschungsgemeins- chaft. References DUTRILLAUX B., 1979; Chromosomal evolution in primates: tentative phylogeny from Microcebus murinus prosimian to man. Hum. Genet., 48: 251-314. JAUCH A., DAI3MER C., LICHTER P., MURKEN J., SCHROEDER-KURTH T. & CREMER T., 1990. Chromosomal COMPARATIVE CHROMOSOME BAND MAPPING IN PRIMATES 71 in situ suppression hybridization o/human gonosomes and autosomes and its use in clinical cytogenetics. Hum. Genet., 85: 145-150. KLEVER M., GROND-GINSBACH C., SCHERTHAN H. & SCHROEDER-KURTH T., 1991. Chromosomal in situ suppression hybridization a/ter Giemsa banding. Hum. Genet., 86: 484-486. LALLEY P.A., DAVlDSON M.T., GRAVES J.A.M., O'BRIEN S.J., WOMACK J.E., RODERICK T.H., CREAU- GOLDBERG N., HILLYARD A.L., DOOLWrLE D.P. & ROGERS J., 1989. Human Gene Mapping 10 - Report o/ the committee on comparative mapping. Cytogen. Cell Gen., 51: 503-532. LENGAUER C., ECKELT A., WEITH A., ENDLICH N., PONELIES N., LIGHTER P., GREULICH K.O. & CREMER T., 1991. Painting o~ defined chromosomal regions by in situ suppression hybridization o~ libraries/rom laser-microdissected chromosomes. Cytogen. Cell Gen., 56: 27-30. LICHTER P., CREMER T., BORDEN J., MANUELIDIS L. & WARD D.C., 1988. Delineation ~ individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization usi g recombinant DNA libraries. Hum. Genet., 80: 224-234. LODECKE, H.J., SENGER G., CLAUSSEN U. & HORSTHEMKE B.,1989. Cloning defined regions o~ the human genome by microdissection f banded chromosomes and enzymatic amplification. Nature, 338: 348-350. O'BRIEN S.J., SEU,~NEZ H.N. & WOMACK J.E., 1988. Mammalian genome organization: an evolutionary view. Ann. Rev. Genet., 22: 323-351. PINKEL D., GRAY J.W., TRASK B., ENCH G. VAN DEN, FUSCOE J. & DEKKEN H. VAN, 1986. Cytogenetic analysis by in situ hybridization with fluorescently labeled nucleic acid probes. C ld Spring Harb. Symp. Quant. Biol., 51: 151-157. PINKEL D., LANDEGENT J., COLLINS C., FUSCOE J., SEGRAVES R., LUCAS J. & GEAY J.W., I988. Fluorescence insitu hybridization with human chromosome specific libraries: Detection o~ trisomy 21 and translocations o~ chromosome 4. Proc. Natl. Acad. Sci. (USA) 85: 9138-9142. SMAI~ M., STANYON R., SMITH D.G. & SINEO L. 1985. High-resolution chromosomes o~ rhesus macaques Macaca mulatta. Am. J. Prim., 9:63-67 STANYON R. & CHIARELLI B. 1983. Phylogeny o~ the hominoidea: the chromosome evidence. J. Human Evol., 11: 493-504. STANYON R., SINEO L., CHIARELLI B., CAMPERIO-CIANI A. HAIMOFF A.R. & MOOTNICK E.H., 1987. Banded karytypes of the 44-chromosome- Gibbons. Folia Primatol, 48: 56-61. STANYON R., ROMAGNO D,, WIENBERG J. & MAURER U., 1990. Sequence o~ DNA replication in Macaca /uscata chromosomes: An outgroupe for phylogenetic comparison between man and pes. Genetica, 80: 45-52. WIENBERG J., JAUCH A., STANYON R. & CREMER T., 1990; Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization. Gen mics, 8, 347-350. YUNIS J.J. & PRAKASH O., 1982. The origin of man: A chromosomal pictor~l egacy. Science, 215: 1525- 1530. Received: Dec. 10 th 1990. Accepted: Dec. 20 th 1990. 

Banded karytypes of the 44-chromosome- Gibbons. Folia Primatol,

Chromosomal evolution in primates: tentative phylogeny from Microcebus murinus prosimian to man.

Chromosomal in situ suppression hybridization a/ter Giemsa banding.

Cytogenetic analysis by in situ hybridization with fluorescently labeled nucleic acid probes. Cold Spring Harb.

Delineation o~ individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries.

High-resolution chromosomes o~ rhesus macaques Macaca mulatta.

HORSTHEMKE B.,1989. Cloning defined regions o~ the human genome by microdissection of banded chromosomes and enzymatic amplification.

Human Gene Mapping 10 -Report o/ the committee on comparative mapping.

Mammalian genome organization: an evolutionary view.

Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization.

Painting o~ defined chromosomal regions by in situ suppression hybridization o~ libraries/rom laser-microdissected chromosomes.

Sequence o~ DNA replication in Macaca /uscata chromosomes: An outgroupe for phylogenetic comparison between man and apes.

situ suppression hybridization o/human gonosomes and autosomes and its use in clinical cytogenetics.

The origin of man: A chromosomal pictor~l legacy.

Comparative chromosome band mapping in primates byin situ suppression hybridization of band specific DNA microlibraries

Universität München: Elektronischen Publikationen

H U M A N  E V O L U T I O N  Vol. 6 - N. 1 (67-71) - 1991 
C. Lengauer, 
J. Wienberg & 
T. Cremer 
Institut [iir Humangenetik und 
Anthropologie, Universitdt 
Heidelberg, Germany 
H.J. Li~decke & 
B. Horsthemke 
Institut fiir Humangenetik, 
Universitdtsklinikum Essen, 
Germany 
Comparative chromosome band mapping 
in primates by in sito suppression 
hybridization of band specific DNA 
microlihraries 
A DNA-library established from microdissected bands 8q23 to 
8q24.1 of normal human chromosomes 8 (Liidecke et al., 1989) was 
used as a probe for chromosomal in situ suppression (CISS-) hybridi- 
zation to metaphase chromosomes of man and primates including 
Hylobates far and Macaca fuscata. Comparative band mapping as first 
applied in this study shows the specific visualization of a single 
subchromosomal region in all three species and thus demonstrates 
that synteny of the bulk sequences of a specific human chromosome 
subregion has been conserved for more than 20 million years. 
Introduction 
Comparisons of chromosome morphology are widely used for phylogenetic recon- 
structions. Fixation of chromosomal changes are rare events and identical chromosomal 
rearrangements in different species are considered to reflect a common evolutionary path. 
Accordingly, ancestral versus derived traits can be established. With few exceptions 
comparative cytogenetics has indicated a highly conserved chromosome morphology 
during primate evolution (DuTRmLAUX, 1979; YUNIS & PRAKASH, 1982; STANYON & 
CHIARELLI, 1983; O'BRIEN et al., 1988). It is widely accepted that chromosome bands 
whose homology has been suggested on the basis of similar banding patterns reflect 
synteny of DNA sequences. However, comparative mapping of individual DNA sequences 
(LALLEY et al., 1990) so far has provided only limited data to unequivocally support this 
hypothesis. These studies are very time consuming and the mapped sequences only reflect 
a very small part of a given chromosome band. 
Recently, we have introduced a new strategy to analyze chromosomal evolution in 
primates based on chromosomal in situ suppression (CISS-) hybridization (WIENBERC et 
al., 1990). A series of bacteriophage DNA-libraries established from flow sorted human 
chromosomes was used to visualize homologies of primate chromosomes directly at the 
DNA level. However, these chromosome specific DNA libraries did not permit to 
reconstruct intrachromosomal rearrangements which are supposed to be prominent in the 
evolution of hominoid karyotypes (DuTRILLAUX, 1979; YUNIS & PRASHAK, 1982; STANYON 
& CHIARELLI, 1983). An unequivocal demonstration of the homology of individual bands 
could be based on CISS-hybridization of band specific DNA libraries. Construction of 
such libraries has become possible by microdissection and microcloning (LODECKE et al., 
1989). 
Here, we have used a micro-DNA-library established from the distal third of band 
8q23 and the proximal two-thirds of sub-band 8q24.1 from normal human chromosomes 
(LODECKE et al., 1989) for comparative band mapping of human and primate species. For 
this comparison we have chosen Hylobates lar and Macaca fuscata in the present study. Due 
to the rapid chromosomal evolution in the lesser apes, it has been exceedingly difficult to 
establish chromosome homologies both between particular gibbon species and other 
9 Edltrice I1 Sedicesimo - Firenze ISSN 0393-9375 
68 LENGAUER, WIENBERG, CREMER, LLIDECKE & HORSTHEMKE 
primates by conventional banding techniques. In contrast, Macaca fuscata shows a much 
higher degree of chromosomal conservation (STANYON & CHIARELLI, 1983). In particular, 
CISS-hybridization of a DNA-library from sorted human chromosomes 8 has shown that 
this chromosome is rearranged in Hylobates lar but has been maintained intact in Macaca 
fuscata (our unpublished data). 
Materials and Methods 
DNA probes and probe labeling: The microlibrary from chromosome bands 8q23- 
8q24.1 was established and characterized by Liidecke et al. (1989). Briefly, 37 dissected 
chromosome fragments were digested with RsaI and ligated to a SmaI-cut pUC-plasmid. 
The inserts were than amplified by PCR using the universal M13/pUC sequencing and 
reverse sequencing primers. In our experiments 100 ng of amplified inserts was again 
amplified in a 100 ul assay containing 1 uM primer each (see above), 10 ~tM Tris-HC1 (pH 
8.4), 50 ~tm KC1, 1.5 ~tM MgC12, 0.001% gelatin, 200 0M of dATP, dGTP, dCTP each, 
100 0M of dTTP, 150 eM bio-ll-dUTP and 2.5 units of Thermus aquaticus DNA 
polymerase. After initial denaturation at 96~ for 3 rain 25 cycles of PCR were carried 
out with denaturation at 96~ for 1 min, annealing at 47~ for 1 min and extention at 
72~ for 2 min. 10 sec. 
Chromosome preparations: Metaphase chromosome spreads for in situ hybridization 
experiments were prepared and stored as described by JAUCH et al. (1990). Human 
chromosomes were derived from phytohemagglutinin (PHA) stimulated peripheral lym- 
phocytes. Gibbon chromosomes (Hylobates lar) were prepared from a lymphoblastoid cell 
line kindly provided by T. Ishida (Primate Center, Kyoto, Japan). The karyotype of this 
cell line was found to be normal diploid (H.lar karyotype: 2n= 44; R. Stanyon, personal 
communication). Macaca fuscata chromosome spreads from primary kidney cultures (STAN- 
YON et al., 1990) were kindly provided by R.Stanyon (Istituto di Antropologia, Genoa, 
Italy). 
Chromosome banding: Chromosome banding previous to CISS-hybridization was 
according to Klever et al. (1991). After routine GTG-banding metaphases were photo- 
graphed (Agfa-Ortho 25), destained with fixative (methanol/acetic acid 3:1) and postfixed 
with 4% formaldehyde (Merck) in PBS for 15 min. Postfixation was essential to preserve 
chromosome morphology in the subsequent CISS-hybridization experiments. 
CISS-hybridization: Sixty ng of PCR labeled probe was used for CISS-hybridization 
(LIcHTER et al., 1988; PmKEL et al., 1988). Biotin labeled probes were detected with 
fluoresceine-conjugated avidine. Signals were amplified once as described by PINKEL et al. 
(1986) and slides were analysed with a Zeiss photornicroscope III equipped for FITC 
fluorescence. Photographs were taken on AGFA-CHROME 1000 RS or Kodak T-MAX 
black and white (400 ASA) films. 
Results 
CISS-hybridization of the microlibrary to human lymphocyte metaphase spreads 
resulted in a complete delineation of band 8q23 and a partial delineation of band 8q24 
(Figure la, b). The telomeric band 8q24.3 clearly remained unstained (Figure 2a, b). This 
Figure 1 - CISS-hybridization of a microlibrary derived from human chromosome bands 8q23-8q24.1 to a 
human metaphase spread, a) GTG-banding, b) FITC-detection of hybridized microlibrary sequences. 
Arrows indicate the labeled subregion on chromosome 8 (bands 8q23-24), arrowheads point to a weak 
hybridization signal on chromosome 9 (band 9q33). 
Figure 2 - GTG-banding, CISS- 
hybridization patterns of the mi- 
crolibrary and idiograms of la- 
beled chromosomes in man 
(HSA) (a-c), Hylobates lar (HLA) 
(d-f) and Macaca ]uscata (MFU) 
(g-i). The Iabeled regions estab- 
lished from a series of chromo- 
somes are indicated in the idio- 
grams by black dots. 
70 LENGAUER, WIENBERG, CREMER, LUDECKE & HORSTHEMKE 
result is in agreement with the molecular characterization of the microlibrary as previously 
described (LODECKE et aL, 1989). Unexpectedly, however, an additional faint signal was 
also evident on chromosome 9q33 (see Discussion). 
In Hylobates lar and Macaca fuscata a single chromosome subregion was delineated 
(Figure 2e, h), while site specific signals on other chromosomes could not be detected. The 
chromosomes with the hybridized subregions were identified on the basis of their G- 
banding pattern as chromosome 9 in Hylobates lar (Figure 2d) according to the idiogram 
published by STANYON et aL, (1987), and as chromosome 8 in Macaca [uscata (Figure 2g) 
according to the numbering system of SMALL et aL (1985). 
Discussion 
CISS-hybridization of DNA microlibraries established from microdissected chromo- 
some regions can be used as a rapid tool to confirm the origin of the bulk DNA sequences 
from the respective region (Lengauer et al., 1991). This study demonstrates the specific 
visualization of single chromosome bands using a human microlibrary established after 
microdissection of bands 8q23-8q24.1. The faint labeling of the additional band 9q33 may 
be explained by an occassional error in the classification of banded chromosomes used for 
microdissection. Alternatively, cross-hybridization may indicate the presence of conserved 
sequences in both bands. This latter explanation seems unlikely, since the decoration of 
complete chromosomes 8 by CISS-hybridization with DNA-libraries established from 
sorted human chromosomes 8 did not result in any detectable cross-hybridization events 
on chromosome 9 and vice versa (our unpublished data). 
CISS-hybridization of the microlibrary to a single subchromosomal region in Hylo- 
bates lar and Macaca [uscata, respectively, demonstrates that synteny of the bulk sequences 
of human chromosome bands 8q23 to 24.1 has been conserved for more than 20 million 
years. Comparative band mapping as first demonstrated in this study provides new 
possibilities for cytotaxonomy. As compared to the physical mapping of individual DNA 
sequences, evolutionary conservation of chromosome bands as well as chromosomal 
rearrangements with breakpoints in specific bands can be rapidly ascertained. Single copy 
probes established from such microlibraries may then be used for more detailed studies of 
possible breakpoints pinpointed within the chromosomal region defined by a microlibrary. 
Acknowledgements 
This work was supported in part by the Deutsche Forschungsgemeinschaft. We would like to thank 
R.Stanyon & T.Ishida for providing us with the primate samples, U.Claussen & G.Senger, Erlangen, for 
collaboration, and A.Wiegenstein for photographic work. C.L. was the recipient of a scholarship from the 
Konrad-Adenauer-Stiftung and T.C. of a Heisenberg-Stipendium from the Deutsche Forschungsgemeins- 
chaft. 
References 
DUTRILLAUX B., 1979; Chromosomal evolution in primates: tentative phylogeny from Microcebus murinus 
prosimian to man. Hum. Genet., 48: 251-314. 
JAUCH A., DAI3MER C., LICHTER P., MURKEN J., SCHROEDER-KURTH T. & CREMER T., 1990. Chromosomal 
COMPARATIVE CHROMOSOME BAND MAPPING IN PRIMATES 71 
in situ suppression hybridization o/human gonosomes and autosomes and its use in clinical cytogenetics. 
Hum. Genet., 85: 145-150. 
KLEVER M., GROND-GINSBACH C., SCHERTHAN H. & SCHROEDER-KURTH T., 1991. Chromosomal in situ 
suppression hybridization a/ter Giemsa banding. Hum. Genet., 86: 484-486. 
LALLEY P.A., DAVlDSON M.T., GRAVES J.A.M., O'BRIEN S.J., WOMACK J.E., RODERICK T.H., CREAU- 
GOLDBERG N., HILLYARD A.L., DOOLWrLE D.P. & ROGERS J., 1989. Human Gene Mapping 10 - 
Report o/ the committee on comparative mapping. Cytogen. Cell Gen., 51: 503-532. 
LENGAUER C., ECKELT A., WEITH A., ENDLICH N., PONELIES N., LIGHTER P., GREULICH K.O. & CREMER 
T., 1991. Painting o~ defined chromosomal regions by in situ suppression hybridization o~ libraries/rom 
laser-microdissected chromosomes. Cytogen. Cell Gen., 56: 27-30. 
LICHTER P., CREMER T., BORDEN J., MANUELIDIS L. & WARD D.C., 1988. Delineation o~ individual human 
chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant 
DNA libraries. Hum. Genet., 80: 224-234. 
LODECKE, H.J., SENGER G., CLAUSSEN U. & HORSTHEMKE B.,1989. Cloning defined regions o~ the human 
genome by microdissection of banded chromosomes and enzymatic amplification. Nature, 338: 348-350. 
O'BRIEN S.J., SEU,~NEZ H.N. & WOMACK J.E., 1988. Mammalian genome organization: an evolutionary 
view. Ann. Rev. Genet., 22: 323-351. 
PINKEL D., GRAY J.W., TRASK B., ENCH G. VAN DEN, FUSCOE J. & DEKKEN H. VAN, 1986. Cytogenetic 
analysis by in situ hybridization with fluorescently labeled nucleic acid probes. Cold Spring Harb. 
Symp. Quant. Biol., 51: 151-157. 
PINKEL D., LANDEGENT J., COLLINS C., FUSCOE J., SEGRAVES R., LUCAS J. & GEAY J.W., I988. 
Fluorescence in situ hybridization with human chromosome specific libraries: Detection o~ trisomy 21 and 
translocations o~ chromosome 4. Proc. Natl. Acad. Sci. (USA) 85: 9138-9142. 
SMAI~ M., STANYON R., SMITH D.G. & SINEO L. 1985. High-resolution chromosomes o~ rhesus macaques 
Macaca mulatta. Am. J. Prim., 9:63-67 
STANYON R. & CHIARELLI B. 1983. Phylogeny o~ the hominoidea: the chromosome evidence. J. Human Evol., 
11: 493-504. 
STANYON R., SINEO L., CHIARELLI B., CAMPERIO-CIANI A. HAIMOFF A.R. & MOOTNICK E.H., 1987. 
Banded karytypes of the 44-chromosome- Gibbons. Folia Primatol, 48: 56-61. 
STANYON R., ROMAGNO D,, WIENBERG J. & MAURER U., 1990. Sequence o~ DNA replication in Macaca 
/uscata chromosomes: An outgroupe for phylogenetic comparison between man and apes. Genetica, 80: 
45-52. 
WIENBERG J., JAUCH A., STANYON R. & CREMER T., 1990; Molecular cytotaxonomy of primates by 
chromosomal in situ suppression hybridization. Genomics, 8, 347-350. 
YUNIS J.J. & PRAKASH O., 1982. The origin of man: A chromosomal pictor~l legacy. Science, 215: 1525- 
1530. 
Received: Dec. 10 th 1990. Accepted: Dec. 20 th 1990. 


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Comparative chromosome band mapping in primates byin situ suppression hybridization of band specific DNA microlibraries

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