Detected by in Situ Digestion with Restriction Endonucleases

Heredity 62 (1989) 269-277 The Geneticat Society of Great Britain Received 19 October 1988

Heterochromatin heterogeneity in Oedipoda germanica (Orthoptera) detected by in situ digestion with restriction endonucleases

C. Lopez-Fernandez Unidad de Genética. Departamento de BiologIa. C- J. Gosalvez and XV, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain. R. Mezzanotte* *lstitutodi Biologia Generale, Facoltá de Medicina e Chirurgia Via Ospedale 121, Cagliari, Italia. Metaphase chromosomes of the grasshopper Oedipoda germanica were treated with Alul, Dde!, HaeIII, Hinfi, HpaH, MboI, MspI, Sau3A and TaqI restriction endonucleases (REs) and subsequently stained with Giemsa. C-banding and fiurochromes of different base pair specificities (DA-DAPI or CMA3) were also used to localize and characterize the heterochromatin. Results indicate that (a) the large blocks of paracentromeric heterochromatin are heterogeneous within each chromosome. This heterogeneity is homogeneously distributed among almost all chromosomes of the genome. (b) The use of REs on fixed chromatin gives rise to a banding pattern not completely correlated with that obtained with C-banding; (c) heterochromatin associated with active NORs displays distinct characteristics: GC-rich (positive CMA3), high frequency of the restriction site CC GG, specific for MspI and HpalI, and low levels of methylation in the internal cytosine.

INTRODUCTION shown in Arcyptera tornosi where at least six types have been detected (Gosálvez et a!., 1987). Constitutiveheterochromatin has been defined as Different cytogenetic tools have made possible permanently condensed blocks of chromatin the characterization of heterochromatic regions located in discrete regions of homologous chromo- within a given genome. Standard C-banding is somes of a given complement (Brown, 1966). Fur- useful for localizing constitutive heterochromatic ther analyses have corroborated its structural and regions in both interphase and metaphase chromo- functional differentiation when compared to the somes (Sumner, 1972), while specific fluro- so-called euchromatin. Heterochromatic regions chromes such as 4'-6-diamidinophenylindole are commonly, though not always, composed of (DAPI), Hoechst 33258, or chromomycin A3 highly repeated DNA sequences, sometimes con- (CMA3) are capable of revealing the relative base taining satellite DNA (for review see Brutlag, pair richness (AT- or GC-rich DNA) of some 1980). On the other hand, the protein component C-bands on fixed metaphase chromosomes may also be particular since peculiar proteins are (Schweizer, 1976). Moreover, particular classes of capable of binding peculiar DNA base sequences GC- or AT-rich DNA can be studied by digesting (Hsieh and Brutlag, 1979). In addition to these fixed chromosomes with restriction endonucleases common features, distinct techniques of induced (REs) (Miller et a!., 1984; Mezzanotte, 1986; longitudinal differentiation of chromosomes Gosalvez eta!., 1987). Extensive cleavage of a given (banding techniques) demonstrate both the quanti- repeated polynucleotide is produced whenever the tative and qualitative heterogeneity of constitutive recognition site of a certain RE is present heterochromatin. For instance, quantitative vari- throughout the structure of such a DNA sequence ations have been reported for heterochromatic and provokes pale or null staining in the chromo- regions of human chromosomes 1, 9, 16 and Y some areas containing such sequences. In contrast, (Podugolnikova, 1979), while qualitative vari- failure of enzyme cleavage occurs whenever the ations derive from the definition of different classes repeated DNA does not contain the enzyme rec- of heterochromatin within the same genome, as ognition site. In the latter case, the staining of these 270 C. LOPEZ-FERNANDEZ, J. GOSALVEZ AND FL MEZZANOTTE regions is similar to that shown by untreated Table 1Different restriction endonucleases used in this work chromosomes. with the sequence cleaved. Dots over C and A indicate Most acridoid grasshoppers have appreciable methylation of these bases quantities of heterochromatin in their comple- Sequence cleaved Sequence not cleaved ments. Results so far obtained by in situ hybridiz- Enzyme ation techniques suggest that such regions display Alul AG!CT some molecular heterogeneity although they are Dde! C!TNAG Haelll GG!CC quite homogeneous when occuppying similar Hinfi G!ANTC regions of non-homologous chromosomes. For HpaII CCGG C!CGG instance, in Atractomorpha similis all distal C- MboI !GATC !GATC bands contain tandem blocks of highly repeated Mspl C!CGG C!CGG DNA, originating from the same family of sequen- Sau3A !GATC ces, which in turn are rather different from the Taqi T!CGA C-bands localized near the centromeres (John et a!., 1986). Such results are also found when scrutinizing base pair composition with fluoro- 30 units of each enzyme. All REs, except Taqi, chromes (John et aL, 1985). were incubated overnight at 37°C. Incubation with In this paper we analyze the extent of TaqI was at 65°C overnight. The same times and heterochromatic molecular heterogeneity in the temperatures were employed in control experi- complement of 0. germanica by in situ selective ments using only the incubation buffer. Staining digestion with REs. Large paracentromeric C-posi- was performed with two per cent Giemsa in phos- tive blocks which characterize this grasshopper are phate buffer for 15 to 30mm. studied to investigate (1) whether they react similarly to digestion with different REs, (2) the arrangement of DNA sequences within these RESULTS blocks and (3) the equilocal distribution of the different heterochromatic fractions in the karyo- Thechromosome complement of 0. germanica type of this species. is 2n =22+X in the male and 2n=°22+XX in the female. All chromosomes have apparently ter- minal centromeres and have been numbered in de- MATERIALSAND METHODS creasing size order, according to their content of euchromatin, into two long (L1-L2), seven medium Meioticchromosomes were obtained from males (M3-M9) and two short chromosome pairs (S10- of 0. germanica collected in Cala Domestica (Sar- S11), the X being a medium sized chromosome. denya). Testes were removed and fixed in The heterochromatin pattern, defined by C- ethanol: acetic acid (3:1).Preparations of mitotic banding is basically restricted to the paracen- chromosomes were obtained from gonial mitosis. tromeric regions of all chromosomes in the form Single follicles of testes were squashed in a drop of large blocks (fig. 1(a), (b)). Autosome L2 is an of 45 per cent acetic acid. The coverstip was exception and shows pale blue chromatin in this removed with a razor blade after immersing the region with small dots positively stained near the slides in liquid nitrogen. Slides were then air dried. centromere. Fluorescence patterns were obtained using the A special chromosome pair, termed megameric, methodology developed by Schweizer (1976, which displays early condensation in meiosis as 1980). Photographs of CMA3 and DA-DAPI bands compared to all remaining autosomes is present in were taken on Valca F-22 film using a ZEISS many species of Orthoptera. In 0. germanica this photomicroscope. Silver impregnation was carried chromosome is the M9 and the Ag-NOR technique out according to the method developed by De Ia localizes the only active nucleolar organizer region Vega and Gosálvez (1983). Nine restriction in the genome (fig. 1(e)) to an interstitial position endonucleases (REs) (see specifications in table on this chromosome (fIg. 1(f)). 1) were employed to digest fixed chromosomes in DA/DAPI and CMAI were used to differenti- fresh air dried preparations. Tandem digestion ate mitotic chromosomes as well to allow some with isoschizomers Mbol-Sau3A and HpaIl-MspI determination of the base composition of the DNA were used to test possible differences in the levels in C-hands (AT- or GC-rich DNA respectively). of methylation of the DNA. The slides were incu- The paracentromeric C-bands showed bright bated with 100 microlitres of incubation buffer plus fluorescence with DA-DAPI (fig. 1(d)). In addi- HETEROCHROMATIN HETEROGENEITY IN OEDIPODA 271

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iy -. Nu 1 - - 4•• ' 4 -' • 4;*1 -. 4 a £ Figure1 (a), (b) C-banded mitotic chromosomes (a) and diplotene (b) showing large blocks of heterochromatin in the paracentromeric regions. (c) Mitotic metaphase stained with CMA3. Note dull differential fluorescence in the centromere regions and more intense bands in chromosome 9 and 6. (d) Same plate stained with DA/DAPI. Differential staining is observed in the paracentromeric heterochromatin. (e), (f) NOR activity. Early melotic prophase showing only a silver precipitate (arrow). Arrowhead corresponds to the X chromosome (e). Selected M9 bivalent showing the localizataion of the single active NOR (f).

tion, there is a weak fluorescence with CMAI in As the results obtained with different REs have small centric regions of all chromosomes (fig. 1(c)). different characteristics, we group our observations There is also a CMA3 positive band interstitially in two main sections: those regarding paracen- located on the megameric M9 chromosome which tromeric regions and those regarding chromo- appears to be at the site of the active NOR (fig. somes areas other than paracentromeric regions. 1(c)). The fluorescence produced by this band is more intense than that found at the centromeres. Paracentromericbands Accordingly a gap is present in this region when chromosomes are stained with DA-DAPI (fig. AllREs used produce longitudinal differentiation 1(d)). One of the medium chromosomes, probably in the complement of 0. germanica but to different the M6, reveals a subdistal CMA3 band in addition extents. Alul, DdeI, MboI, Sau3A, (fig. 2) and to a centric and an interstitial band which seem to TaqI (fig. 4(a), (c)) produce clearcut chromosome include a large DA-DAPI heterochromatic band differentiation and high contrast between eu- and (compare fig. 1(c) and 1(d)). heterochromatin. On the contrary, HaeIII, HpaII, MspI and Hinfi induce low contrast between eu- and heterochromatin (fig. 3(a), (c)). Restrictionenzymes Most REs produce paracentromeric bands Insitu digestion of mitotic chromosomes with REs which correspond to those obtained with C- and subsequent staining with Giemsa produces banding, although MboI and Sau3A reveal only a linear bands which are different, at least in some small positive band in that region of chromosome chromosomes, from those obtained with C- S10 (fig. 2(b), (d)). Interestingly, Taqi produces a banding. reverse pattern in this chromosome with respect 272 C. LOPEZ-FERNANDEZ, J. GOSALVEZ AND R. MEZZANOTTE

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Figure 2 (a) Selective digestion with Alul. Arrows show positive bands not obtained with C-banding or lluorochromes. (b) Selective digestion with Mbol. Arrowed chromosomes indicate bands not obtained with C-handing. Note the selective digestion produced in the paracentromeric heterochromatin of the S10 chromosome. The same pattern is obtained with the isoschizomer Sau3A (d). (c) Selective digestion with DdeI. to that obtained with MboI and Sau3A. That is, general behaviour of the paracentromeric hetero- the only prominent paracentromeric positive band chromatin in metaphase chromosomes is shown in is located on chromosome S10 (fig. 4(c)). Other fig. 5 using the L1 chromosome. While C-bands REs which produce C-like bands such as Alul or and Alul localize constitutive heterochromatin, DdeI show a similar pattern on S to that obtained DA/DAPI and CMA3 reveal the bipartite nature with C-banding (fig. 6). Furthermore DA/DAPI of this zone. Taqi does not digest all the DA/DAPI and CMA3 show a bipartite nature to this region or Alul positive bands. However, the TaqI band in terms of AT- or GC-rich DNA (fig. 6). does not correspond to that obtained with CMA3. Taqi produces multiple positive bands interspersed with unstained regions within paracentromeric heterochromatin. This effect is clearly vis- Non-paracentromericbands ible in early prophase chromosomes (fig. 4(a)). This differential intra-heterochromatic digestion is Togetherwith the paracentromeric bands only partially observed in metaphase chromo- described above several REs produce some posi- somes (see multiple arrowed chromosomes in fig. tive bands as well as negative gaps in other chromo- 4(c)). The visual effect produced by Taqi in meta- some regions. HpaII and MspI induce unstained phase chromosomes is a reduction in the size of gaps in an interstitial region of S9 chromosome the stained band in most of the chromosomes (fig. (fig. 3(c), (d), (e)). These gaps are particularly 4(c)). All remaining enzymes induce homomorphic evident in condensed chromosomes and corre- bands both in early prophase (compare fig. 4(a) spond to the chromatin associated to the active with 4(b)) and in metaphase. A comparison of the NOR detected with Ag-staining. This effect is also positive bands areobtainedinthedistal regionof band onchromosomeM5(fig. 2(b),(d)). Sau3A, butnotAlul,induce apositiveinterstitial chromosome M6(fig.2(a), (b),(d)).Mboland same enzymesrevealalarge interstitialbandin with Alul,Sau3AandMboI (fig.2(b),(d)).The (see arrowsinfig.2(a))areobserved afterdigestion (fig. 3(a)). observed inchromosomesdigestedwithHaeIII Figure HETEROCHROMATIN HETEROGENEITYINQEDIPODA Finally, inslides treatedwithTaqi,discrete Distal positivedotsindifferentchromosomes chromosomes withMspI(d)andHpall(e).Notethepresenceofgaps inchromosomeM9. is produced.(c)SelectivedigestionwithMspI.Noteagapinchromosome M9.(d),(e)Selectivedigestionofmetaphase 3 (a)Selectivedigestionwithl-laelII.NoteagapinchromosomeM9. (b)SelectivedigestionwithHinfl.Nodifferentiation a Se. a F 1\ 1)

b p a 'I results (i)confirm thecapabilityofREs todigest (for review, see BabuandVerma,1987). Our somes afterdigestionwith REshaveappeared terization ofheterochromatin infixedchromo- During DISCUSSION some M5(fig.4(c)). S9 andthesubparacentromericregionofchromo-

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Figure4 (a) Mitotic prophase digested with Taqi. Arrows indicate the intra-heterochromatin banding produced by a differential digestion. (b) Mitotic prophase digested with Mu!. Note homogeneous staining in all the centromere regions. (c) Metaphase plate digested with TaqI. The digestion of the paracentromeric heterochromating produces diminished staining in those regions. Arrows on numbered chromosomes indicate bands not obtained with C-banding. Arrows on unnumbered chromosomes indicate the metaphase visualization of the intra-heterochromatic digestion. HETEROCHROMATIN HETEROGENEITY IN QEDIPODA 275

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Figure5 Selected L1 chromosomes to show the differential reaction of the heterochromatin to different treatments. Arrows indicate the extent of the differential staining.

the chromatin whatever its characteristics of con- paracentromeric areas of chromosomes 1, 9 and densation, (ii) reveal the existence of certain 16 are characterized as highly condensed chromosome regions possibly containing repetitive chromatin (heterochromatin) containing satellite DNA sequences though they are not detected with DNA sequences. In both cases these regions result conventional banding techniques and (iii) uncover digested by enzymes such as Bst NI and Hinil, the heterogeneity of equilocally distributed hetero- respectively (Southern, 1975; Miller et a!., 1984; chromatin. Bianchi et a!., 1985). Such specific efficiency is Data so far obtained indicate that some REs shown in 0.germanicaby the different patterns are able to selectively digest chromosomal DNA of bands obtained on highly packed paracen- regardless of the accessibility of the target, i.e., tromeric heterochromatin with REs such as Alul REs are able to recognize and cut specific targets or TaqI, but is also supported by the production whatever the nature of chromatin compactness. of a unique and specific band on M5 chromosome For instance, all mouse centromeres and human after the treatment with the isoschizomeres MboI

Figure 6 Selected S10 chromosomes to show the differential reaction of the heterochromatin to different treatments. Arrows indicate the extent of the differential staining. 276 C. LOPEZ-FERNANDEZ, J. GOSALVEZ AND B. MEZZANOTTE or Sau 3A. All these facts indicate that REs are paracentric heterochromatin and clustered in adja- capable of acting on chromatin whatever its struc- cent but not contiguous blocks. On the contrary, ture, despite the presumable differences in the homogeneous positive staining obtained in the molecular structure of distinct enzymes with iden- same regions with enzymes such as Alul, DdeI, tical targets. The same arguments apply to other Sau3A, MboI and HaeiII indicates the absence of enzymes which cut identical or similar targets other restriction sites in the different subunits form- (HaeIII, MspI and HpaII, in our case). In fact the ing the heterochromatic blocks, at least with gap produced by MspI and HpaII in the NOR- sufficient frequency to produce removable DNA associated CMA3 band suggests two important fragments. characteristics of this particular chromosome These equilocal patterns of heterochromatin region: (i) the massive presence of CC GG restric- also have some remarkable exceptions. For in- tion sites which agrees with the high GC richness stance, Sau3A and MboI produce massive diges- of the DNA according to the strong positive tion of the paracentromeric heterochromatin of fluorescence obtained with CMA and (ii) the chromosome S10 probably due to the high absence or minor incidence of methylated frequency of the specific target which permits cytosines (both internal and external since both massive cleavage and removal of the DNA. The REs produce the same banding pattern). differential arrangements of the chromatin in this Constitutive heterochromatin is usually related paracentromeric heterochromatin is reinforced by to C-banded chromosome regions which are the homogeneous staining obtained after the diges- usually those differentially fluorescing with base- tion with Taqi. The restriction sites in this zone pair specific dyes (John et a!., 1985; Schmid et a!., are clearly different in number and position to 1986). In general, this extends to the banding pat- those found in other chromosomes. terns obtained with certain REs (Lloyd and Thor- gaard, 1988). However, as reported in the grasshopper A. tornosi (Gosalvez et a!., 1987), complete Acknowledgements We thank Dr C. Garcia de Ia Vega for correspondence between C-bands and chromo- his very helpful comments and Dr J. L. Bela for technical somal areas differentially reacting to the RE treat- assistance. This research was partially supported by CICYT ment is not always found. In 0. germanica, as in (PB 86-0106)andAcciones Integradas Hispano-Italianas A. lornosi, digestion with Alul induces telomeric (1988). dots in some chromosomes. These regions do not show C-bands or bright fluorescence. The interstitial band of chromosome M5 is not C-banded REFERENCES and is only observable when digesting chromosomes with Sau3A or MboI. These results confirm BABU.A. AND VERMA, R. S. 1987. Chromosome structure: Euchromatin and Heterochromatin. mt. Rev. Cytol., 108, that C-bands do not necessarily coincide with RE- 1-49. bands and also suggest that repetitive DNAs BlANCH!, M. S., BlANCH!, N. 0., PANTELIAS, G.E. AND WOLFF. sequences are probably included in those bands. S. 1985. The mechanisms and pattern of banding induced The "equilocal distribution of hetero- by restriction endonucleases in human chromosomes. Chromosoma, 91, 131—136. chromatin" hypothesis predicts that certain BROwN. S. w. 1966. Heterochromatin. Science, 151, 417—425. chromosome regions within all the members of a BRUTLAG, ID. L. 1980. Molecular arrangement and evolution of given complement tend to accumulate this kind of heterochromatic DNA. Ann. Rev. Gene!. 14, 121-144. chromatin (Heitz, 1933; Schweizer and Loidi, DE LA VEGA, C. AND GOSALVEZ. .i. 1983. Differential NOR 1987). This has been confirmed by several reports activity in two acridoid species. Biol. Zbl., 102, 545-549. GOSALVEZ, J., BELLA, J. L., LOPEZ-FERNÁNDEZ, C. AND that also demonstrate the molecular heterogeneity MEzzANOTm, P.. 1987. Correlation between constitutive of heterochromatin located in different chromo- heterochromatin and restriction enzyme resistant some regions (Schweizer and Ehrendorfer, 1983; chromatin in Arcyptera tornosi (Orthoptera). 1-teredity, 59, John et cii.,1985).Our results in 0. germanica show 173—180. HEITZ, E. 1933. Die somatische heteropyknose bei Drosophila homogeneously C-banded large paracentromeric melanogaster und ihre genetische bedeutung. Z. Zellforsch, heterochromatic blocks within each chromosome. 20, 417-425. However specific fluorochromes reveal a bipartite HSIEH, T. AND BRUTLAG, D. L. 1979. A protein that preferen- structure of these blocks (heterogeneity). Similarly tially binds Drosophila satellite DNA. Proc. Nati. Acad. TaqI produces an interspersed pattern of positive Sci. USA, 76, 726—730. JOHN, B., APPELS. R. AND CONTRERAS, N. 1986. Population and dull bands within these C-bands in most of cytogenetics of Atractomorpha ,cimilis.II. Molecular the chromosomes. This arrangement suggests that characterization of the distal C-band polymorphisms. repeated sequences are equilocated, in general, in Chromosoma, 94, 45-58. HETEROCI-IROMATIN HETEROGENEY IN QEDIPODA 277

JOHN. B., KING, M., SCHWEIZER, D. AND MENDELAK, M. 1985. SCI-IWEIZER, D. 1976. Reverse fluorescent chromosome band- Equilocalityof heterochromatin distribution and hetero- ing with chromomycin and DAPI. Chromosoma, 58, 307- chromatin heterogeneityinacrididgrasshoppers. 324. Chromosoma, 91, 185-200. SCHWEIZER, ID. 1980. Simultaneous fluorescent staining of R LLOYD, M. AND TI-IORGAARD, G. H. 1988. Restriction endo- bands specific heterochromatic regions (DA-DAPI bands) nuclease banding of rainbow trout chromosomes. in human chromosomes. Cytogenet. Cell. Genet., 27,190- Chromosoma, 96,171-177. 193. MEZZANOTTE, R. 1986. The selective digestion of polytene and SCUWEIZER, D. AND LOIDL, .r. 1987. A model for hetero- mitotic chromosomes of Drosophila melanogaster by the chromatin dispersion and the evolution of C-bands pat- Alul and HaelIl restriction endonucleases. Chromosoma, terns. Oiromosome Today, Vol. 9, Allen and Unwin. Lon- 93, 249—255. don, pp. 61-79. MILLER, D. A., GOSDEN, J. R., HASTIE, N. D. AND EVANS, SCHWEIZER, D. AND EHRENDORFER, F. 1983. Evolution of H. j.1984.Mechanism of endonuclease banding of chromo- C-band patterns in Asteraceae-Anthemidae. Biol. Zbl., 102, somes. Exp. Cell. Res., 155, 294—298. 637—655. PODUGOLNIKOVA, 0. A. 1979. The quantitative analysis of SOUTHERN, E. M. 1975. Long periodicities in mouse satellite polymorphism of human chromosomes 1,9, 16 andY. III. DNA J. Mo!. Biol., 94, 5 1-69. Study of relationships of C segments length in individual SUMNER, A. T. 1972. A simple technique for demonstrating karyotypes. Hum. Genet., 49, 261—268. centromeric heterochromatin. Exp. Ce!!. Res., 75,304-306. SCHMID, M., HAAF, T., orr, G., SCHERES, J. M. J. C. AND WENING, .i. A. B. 1986. Heterochromatin in the chromosomes of gorilla: characterization with distamycin A/DAPI, D287/170, chromomycin A3, quinacrine and 5- azacytidine. Cytogenet. CelL Genet., 41, 71—82.