|Address: Dr. J. Surrallés. Group of Mutagenesis.
Genetics Unit, Department of Genetics and Microbiology. Edifici Cn, Universitat Autňnoma
de Barcelona. 08193 Bellaterra, Cerdanyola del Vallčs, Barcelona, Spain.
Fax: +343 581
23 86; Phone: +343 581 25 97; E-mail: email@example.com
Keywords: Chromosomes; CpG islands; Gene density; FISH; Repair;
Introduction: from micronuclei to DNA repair
My initiation in the field of Mutagenesis was made possible thanks to my former
professor of Genetics Ricardo Marcos who put his trust in me almost 10 years ago. He was
leading a small research group at the Department of Genetics of the Autonomous University
of Barcelona. This group is nowadays the Group of Mutagenesis, one of the biggest and most
dynamic research teams in Spain. He asked me to read a paper by Michael Fenech describing
the micronucleus (MN) assay in human lymphocytes (Fenech and Morley, 1985). I soon learned
that MN had something to do with Paramecium aurelia also being chromosome
fragments or whole chromosomes that are left behind during anaphase and appear in the
cytoplasm of daughter cells as small additional nuclei. As Ricardo knows very well, my
attempts to break and lose chromosomes, and therefore to induce MN, turned out to be my
life sentence: thousands of hours of microscope, hundreds of blood samples, and too many
hours in front of a computer screen. I even used to dream I was floating inside a giant MN
trying to find out whether it was a chromosome fragment or a misegregating whole
Soon after the beginning of my PhD I discovered that travelling was fun and I started
to practice scientific tourism by visiting Caterina Tanzarella's laboratory in Rome, in
1991. We set up a fruitful collaboration with the aim standardizing the MN assay. During
my stay in Rome, Caterina showed me a nice picture of the first MN after kinetochore
immunolabelling (Degrassi and Tanzarella, 1988). This photograph was the end of my dream
and the beginning of a nightmare since I had to learn extremely complicated protocols to
study the dual origin of MN: fluorescence in situ hybridization (FISH) and
immunocytogenetics. I then moved to Michelline Kirsch-Volders's laboratory where I used to
rush behind Paul van Hummelen while he was doing his first pancentromeric FISH. I gained
more experience with FISH in Marja Sorsa's and Hannu Norppa's laboratory in Helsinki and
in immunocytogenetics with Michael Fenech and Peter Jeppesen in Australia and Scotland,
respectively. My most sincere acknowledgement to all these people that have contributed to
my scientific training.
My trip to Australia was not only tourism. Michael Fenech proposed a simple way to
study DNA repair by converting excision repairable DNA lesions to MN with the use of a DNA
repair inhibitor Ara-C, that blocks the refilling step of excision repair. The unfilled
gaps were then converted to chromosome breaks and, subsequently, to MN after S phase
(Fenech and Neville, 1992). We proved this model by converting UV-light induced excision
repairable DNA lesions to MN and, as hypothesized, all the induced MN were kinetochore
negative and, therefore, derived from chromosome fragments (Fenech et al., 1994). We then
applied this methodology to study chemically induced excision repair (Surrallés et al.
1995). This is the silly way a MN lover was introduced into the field of DNA repair at the
Heterogeneous repair in chromosomes with high and low gene density
As any young scientist interested in breaking and sticking chromosomes, I was following
the steps of a wise man called A.T. Natarajan, Nat. I was really lucky he accepted me to
join his laboratory in the Department of Radiation Genetics and Chemical Mutagenesis in
Leiden, Holland. After a few coffee breaks and late-night beers, Nat completely changed my
concept of science. He had and still has a deep personal and scientific influence on me,
so I focused all my efforts on his main research line: the mechanisms of chromosomal
aberrations and how chromatin structure and DNA repair modulate chromosome fragility.
I initially recovered the MN approach of using DNA repair inhibitors to convert
excision repair unfilled sites to chromosome breaks with the aim of comparing induced
breakage in chromosomes with high and low gene density. The rational of this approach is
straight: gene density reflects the heterogeneity of diverse chromosomes in terms of gene
activity and chromatin structure. Assuming that DNA repair is highly influenced by
chromatin structure and genetic activity, we hypothesized that high gene density
chromosomes would be preferentially repaired and, therefore, more sensitive to the
inhibitory effects of the DNA repair inhibitor.
In order to compare DNA repair in chromosomes with high and low gene density, the next
step was to find out an easy way to "see" the gene density of a given
chromosome. The use of the word "see" was deliberate. Some years ago a Nobel
laureate said that nothing is proved until it is demonstrated at the molecular level. My
problem is that I only believe what I see with my own eyes. This is probably one of the
many reasons why he got the Nobel prize and I will never do. Anyhow we managed to
visualize gene density cytogenetically by using reverse FISH with a probe enriched in CpG
islands that we had isolated from human genomic DNA (Surrallés et al., 1997a). This
approach belongs to Craig and Bickmore (Craig and Bickmore, 1994) and it is based on the
widely accepted assumption that most genes have CpG islands in their regulatory regions.
We digested genomic DNA with the restriction enzyme HpaII cutting CCGG sites. As high gene
density segments are rich in CCGG sites, many tiny fragments are generated by the HpaII
digestion. After 32P end-labelling and electrophoresis, these tiny fragments
can be recovered from the gel, labelled with digoxigenin and used as a probe to paint back
the chromosomes by FISH.
After this procedure, Nat was plethoric when he "saw with his eyes" a
metaphase where some chromosomes were completely banded and some others were not.
Chromosome 18 and 19 appeared to be the ones with the lowest and highest gene density
signal, respectively. Amongst the big chromosomes, we selected chromosome 1 and chromosome
4 as examples of high and low gene density chromosomes, respectively. We then treated
human lymphocytes with the ethylating agent EMS and Ara-C, and analysed induced chromosome
breakage in chromosomes 1,4,18, 19 and 20 by chromosome painting. After correcting for the
DNA content, chromosomes with increasing gene density were increasingly more sensitive to
the inhibitory effect of Ara-C upon the EMS induced repair sites. Considering that
functional base and nucleotide excision repair is required for the clearance of ethylation
damage from DNA (Sitatam et al., 1997), we interpreted our results as showing that
chromosomes with high gene density are preferentially repaired in human cells (Surrallés
et al., 1997a).
Towards a repair karyotype: insights from xeroderma pigmentosum cells
It is well known that nucleotide excision repair (NER) is preferentially directed to
actively transcribed genes and their transcribed strands through transcription coupled
repair mechanisms (Friedberg et al., 1996). Since gene density and activity is clustered
in some chromosomes and chromosomal regions, the next challenge was to visualize those
chromosomal regions subject to preferential repair. These regions should theoretically
co-localize with those regions rich in CpG islands, since chromosomal bands harboring
actively transcribed genes would be expected to be preferentially repaired. The final aim
was to generate what we called a "repair karyotype". This repair karyotype would
allow us to corroborate our observations that chromosomes with high gene density, such as
chromosome 1 or 19, are preferentially repaired when compared to gene poor chromosomes,
such as chromosome 4.
When somebody is fully ignorant about something, the best she/he can do is to seek for
advice, so I suggested the idea of generating a repair karyotype to a world authority in
the field of DNA repair, Leon Mullenders. Considering that Leon was on his feet all day
long talking about DNA repair and that his office was only two meters away from my
laboratory bench, our interaction, as well as my first contact with repair deficient cell
lines, was almost unavoidable.
Xeroderma pigmentosum group C (XPC) cells are known to be partially deficient in NER
since they can only repair the transcribed strands of active genes (Venema et al., 1991).
UV-induced repair patches in confluent XPC cells were labelled with BrdU. After EcoRI
digestion and CsCl gradient, unreplicated DNA fragments containing BrdU-repair patches
were isolated with antibodies against BrdU and an immunomagnetic system which is too long
to explain now (Kalle et al., 1991). The extracted repaired fragments were random primed
with biotin and used as a probe to perform reverse FISH to metaphases from normal cells.
In other words, we mapped repaired DNA by FISH. The chromosomal distribution of FISH
signals was compared to that found with the unrepaired fractions containing non-repaired
bulk DNA and the non-transcribed strands of active genes. To further control the
distribution of repair sites in human chromosomes we also generated similar probes with
wild type cells and xeroderma pigmentosum group A (XPA) cells, which are known to be
completely deficient in NER. Preliminary observations indicated that there are clusters of
transcription coupled repair in early replicating R bands (light G bands) and, therefore,
that overall early replicating chromosomes such as gene-rich chromosomes 1 and 19 are
preferentially repaired when compared to late replicating chromosomes. All avenues seem to
lead to the same conclusion: high gene density chromosomes are preferentially repaired in
The inactive X chromosome and heterochromatin: presenting a model to study
heterogeneous repair at the chromosome level
The specific time interval during DNA-synthesis (S-phase) at which a given DNA sequence
is being replicated is highly associated with the transcriptional activity of that
sequence. Accordingly, expressed DNA loci undergo early replication whereas unexpressed
loci replicate late. A clear example is the inactive X chromosomeand the non-coding
satellite DNA forming the constitutive heterochromatin, which replicate late in S-phase
(see Yeshaya et al., 1998, and references therein). In order to further visualize
heterogeneous repair at the chromosome level we applied the above repair inhibition
approach and interphase FISH with tandem probes to provide visual evidences for a low
level of DNA repair in human constitutive heterochromatin, band 1q12. We speculated that
the observed repair deficiency in 1q12 probably accounts for its fragility and involvement
in cancer chromosomal aberrations (Surrallés et al., 1997b).
In our attempts to find out models to study heterogeneous repair at the chromosome
level, we concluded that the inactive X chromosome had all the features a good model
required. X chromosome inactivation results in silencing of transcriptional activity in
all but one X-chromosome in somatic female cells, hence allowing dosage compensation of
X-linked genes between males and females (Lyon, 1961). In previous studies comparing
euchromatin and heterochromatin with respect to chromosome fragility and repair it was not
possible to know whether it is the heterochromatic state or the heterogeneous genetic
background which actually accounts for the observed differences between heterochromatin
and euchromatin. However, the inactive X chromosome provides us with a unique opportunity
to overcome this limitation as both X chromosomes (active and inactive) have the same
genetic background and, therefore, the active X chromosome serves as internal control to
study the role of transcriptional activity and chromatin conformation in DNA repair.
One of the prerequisites of using the inactive X chromosome as a model to study
intragenomic heterogeneity in DNA repair is that X inactivation is not spread to autosomal
material in the case of translocations X chromosome-autosome. Otherwise, selection against
cells carrying translocations involving the inactive X chromosome would lead to biassed
results. We therefore analyzed de novo induced translocations between the
inactive X chromosome and autosomes to study the spreading of X-inactivation with respect
to the position of the XIST gene This gene is known to control the spreading and
initiation of inactivation during early embryonic development (Brown et al., 1991; Penny
et al., 1996; Herzing et al., 1997; Lee and Jaenisch; 1997). We had to develop new methods
to check for cis-limited spreading of X inactivation in X chromosome-autosome
translocations. Thus, translocations involving any of the two X-chromosomes were detected
by means of FISH with X-chromosome specific red painting probes. The activation status of
the chromosomes involved in the translocation was determined by simultaneous
immunocytogenetics with antibodies either against acetylated histone H4 as a cytogenetic
marker of gene expression (Jeppesen and Turner, 1993; Surrallés et al., 1996) or against
BrdU incorporated at late S-phase as a cytogenetic marker of the late-replicating inactive
X chromosome (Willard and Latt, 1976). Xq13 band carrying the XIST gene was localized by
computer-assisted generation of the DAPI banding pattern. Alternatively, the position in cis
or trans of the XIST gene in the reciprocal products of the translocation was
determined by simultaneous XIST gene specific FISH and computer enhancing. Our study in
differentiated somatic cells provided visual demonstration that the X-inactivation was not
spread to the translocated autosomes irrespective of the position of the XIST gene
(Surrallés et al., 1998a), so the inactive X chromosome could be faithfully used as model
to study heterogeneousDNA repair at the chromosome level.
Accordingly, we induced chromosome breakage in human lymphocytes with X-rays in the
presence or absence of an inhibitor of double strand break repair, adenine 9--d-arabinofuranoside
(Ara-A). Our data surprisingly indicated that both chromosomes are equally radiosensitive.
However, the inactive and highly condensed state of the inactive X chromosome enhanced the
inhibitory effect of Ara-A upon the repair of X-ray-induced breaks. Thus, the observed
fragility is the result of a balance between the actual number of breaks induced in each
chromosome and their differential processing (Surrallés et al., 1998b). The next step
will be to compare the extent of UV-radiation induced NER in the active and inactive X
chromosome. As NER is preferentially directed to actively transcribed regions, we would
expect a lower level of repair in the inactive X chromosome.
Does gene density modulate the persistence of chromosome damage?
Once I had managed to settle back into the Spanish society, I had in mind that, besides
different radiosensitivity and repair, another factor that might modulate interchromosomal
differences in the number of breaks detected after past exposure is a differential
persistence of translocations involving different chromosomes. Low persistence of
translocations might be related to cell lethality as a result of gene truncation or
position effects. If this is true, then one would expect a lower persistence of
translocations involving chromosomes with high gene density. To verify this hypothesis we
are analysing the persistence of translocations involving chromosomes 1 and 19 (with high
gene density) and 4 and 18 (with very low gene density). Translocations were induced by
ionizing radiations in a wild-type lymphoblastoid cell line and samples were collected and
harvested 1, 3, 7, 14, 28, 42 and 56 days after irradiation. After scoring 4000 metaphases
per chromosome and time point (including untreated controls), chromosome aberrations
involving chromosome 1 declined slightly faster than those involving chromosome 4,
suggesting that aberrations involving the gene-rich chromosome 1 are less stable. Further
analysis will allow us to state whether this tendency is biologically relevant and
actually related to gene density.
Back in my home laboratory, I am really lucky my PhD students are continuing the job. I
am trying to pass them all I have learnt from my mentors and teachers so the knowledge
will not fade but, like wine, improve through the years. This is probably the tribute I
can pay to all the people that have made it possible for me to get this Award and that
have put their trust in me over the past 10 years, including my family and my wife. Last
but not least. Thank you all.
This overview is the subject of the EEMS Young Scientist Award 1998 Lecture I gave
during the 28th-EEMS Annual Meeting in Salzburg. I would like to thank the Award Committee
for the prize and for giving me the opportunity to attend the EEMS-Meeting.
Brown, C.J., A. Ballabio, J.L. Rupert, R.G. Lafreniere, M. Grompe, R. Toniorenzi and
H.F. Willard (1991) A gene from the region of the human X-inactivation centre is expressed
exclusively from the inactive X-chromosome. Nature 349: 38-44.
Craig,J.M. and W.A. Bickmore (1994) The distribution of CpG islands in mammalian
chromosomes. Nature Genet. 7: 376-382.
Degrassi, F. and C. Tanzarella (1988) Immunofluorescent staining of kinetochores in
micronuclei: a new assay for the detection of aneuploidy. Mutation Res. 203: 339-345.
Fenech and Morley (1985) Measurement of micronuclei in human lymphocytes. Mutation Res.
Fenech, M. and S. Neville (1992) Conversion of excision repairable DNA lesions to
micronuclei within one cell cycle in human lymphocytes. Environ. Mol. Mutagen. 19: 27-36.
Fenech, M., J. Rinaldi, and J. Surrallés (1994) The origin of micronuclei induced by
cytosine arabinoside and its synergistic interaction with hydroxyurea in human
lymphocytes. Mutagenesis 9: 273-277.
Friedberg,E.C. (1996) Relationships between DNA repair and transcription, Annu. Rev.
Biochem. 65, 15-42.
Herzing, L.B.K., J.T. Romer, J.M. Horn, A. Ashworth (1997) Xist has properties of the
X-chromosome inactivation centre. Nature 386: 272-275 (1997).
Jeppesen, P., and B.M. Turner (1993) The inactive X chromosome in female mammals is
distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene
expression. Cell 74: 281-289.
Kalle, W.H.J., A.-M. Hazekamp-van Dokkum, P.H.M. Lohman, A.T. Natarajan, A.A.van
Zeeland, and L.H.F. Mullenders (1992) The use of streptavidin-coated magnetic beads and
biotinylated antibodies to investigate induction and repair of DNA damage: analysis of
repair patches in specific sequences of uv-irradiated human fibroblast. Anal. Biochem.
Lee, J.T. and R. Jaenisch (1997) Long-range cis effects of ectopic
X-inactivation centres on a mouse autosome. Nature 386, 275-279.
Lyon, M.F. (1961) Gene action in the X-chromosome of the mouse (Mus musculus L.),
Nature, 190, 372-373.
Penny, G.D., G.F. Kay, S.A. Sheardown, S. Rastan, N. Brockdorff (1996) Requirement for
Xist in X-chromosome inactivation. Nature 379: 131-137.
Sitatam, A., G. Plitas, W. Wang and D. A. Scicchitano (1997) Funtional nucleotide
excision repair is required for the preferential removal of N-ethylpurines from the
transcribed strand of the dihydrofolate reductase gene in Chinese hamster ovary cells.
Mol. Cell. Biol. 17: 564-570.
Surrallés J., N. Xamena, A. Creus, and R. Marcos R (1995) The suitability of the
micronucleus assay in human lymphocytes as a new biomarker of excision repair. Mutat Res.
Surrallés, J., P. Jeppesen, H. Morrison, A.T. Natarajan (1996) Analysis of loss of
inactive X chromosome in interphase cells. Am. J. Hum. Genet. 59: 1091-1096.
Surrallés, J. S. Sebastian, and A.T. Natarajan (1997a) Chromosomes with high gene
density are preferentially repaired in human cells, Mutagenesis 12: 437-442.
Surrallés, J., F. Darroudi, and A.T. Natarajan (1997b) Evidence for low level of DNA
repair in human chromosome 1 heterochromatin, Genes Chromosomes and Cancer 20: 173-184.
Surrallés, J. and A.T. Natarajan (1998a) Position effect of translocations involving
the inactive X chromosome: physical linkage to XIC/XIST does not lead to long-range de
novo inactivation in human somatic cells. Cytogenet. and Cell Genet. (in press)
Surrallés, J. and A.T. Natarajan (1998b) Radiosensitivity and Repair of the Inactive X
chromosome. Insights from FISH and Immunocytogenetics. Mutation Res. (in press)
Venema, J., A. Van Hoffen, V. Karcagi, A.T. Natarajan, A.A. van Zeeland, amd L.H.F.
Mullenders (1991) Xeroderma pigmentosum complementation group C cells remove pyrimidine
dimers selectively from the transcribed strand of active genes. Mol. Cell. Biol. 11:
Willard, F.H. and S.A. Latt (1976) Analysis of deoxyribonucleic acid replication in
human X chromosomes by fluorescence microscopy. Am. J. Hum. Genet 28: 213-227.
Yeshaya, J., R. Shalgi, M. Shohat and L. Avivi (1998) Replication timing of the various
FMR1 alleles detected by FISH: inferences regarding their transcriptional status. Human
Genet. 102: 6-14.