Hereditas 119: 179-185 (1993)

Characterization of the heterochromatin of the darkling beetle Misolampus goudoti: cloning of two satellite DNA families and digestion of chromosomes with restriction enzymes JOAN PONS, EDUARD PETITPIERRE and CARLOS JUAN

Laboratori de GenPtica, Departament de Biologia Ambiental, Universitat de les Illes Balears, Palma de Mallorca, Spain

PONS, J., PETITPIERRE, E. and JUAN, C. 1993. Characterization of the heterochromatin of the darkling beetle Misolampus goudoti: cloning of two satellite DNA families and digestion of chromosomes with restriction enzymes. - Hereditas 1 1 9 179-185. Lund, Sweden. ISSN 0018-0661. Received March 10, 1993. Accepted May 14, 1993

The darkling beetle Misolampus goudoti Er. has 58 % of C-banded chromosome material. In this paper we deal with the study of the heterochromatin of this insect both by molecular and cytogenetical methods. Two different satellite DNA families have been characterized in Misolampus goudoti by agarose gel electrophore- sis of EcoRI and PstI restriction fragments, respectively. The EcoRI family is composed of a monomeric unit of 196 bp (64.3 % A-T rich) DNA sequence, representing about 120,000 copies per haploid genome. The presence of frequent intermediate-size satellite variants and an internal direct repetition of 61 bp in the EcoRI repetitive main monomer suggest that the evolution of this satellite proceeded by unequal crossing-over, occurring both within and between the 196 bp unit. Another highly repetitive sequence, defined by digestion of genomic DNA with PstI, has a more complex unit of 1.2 kb with about 70,000 copies per haploid genome. In situ digestion of M. goudoti chromosomes with restriction enzymes shows a non-specific chromosome DNA extraction from pericentromeric positions with EcoRI and chromosome specific extraction of DNA with PstI and HinfI. This is discussed in relation to the chromosomal location of both satellites.

Carlos Juan, Laboratori de GenPtica, Departament de Biologia Ambiental, Uniuersitat de les Ilks Balears, E-07071 Palma de Mallorca, Spain

In the last 20 years or so, an increasing amount of data have been produced on satellite DNA from a variety of eukaryotes. However, the ques- tion of its possible function, if any, and that of the origin of these highly repeated DNA se- quences are still a matter of debate (JOHN and MIKLOS 1979, 1988).

Darkling beetles have been used in several satel- lite DNA studies showing interesting characteris- tics. The satellite DNA sequences of tenebrionids usually represent an important amount, 30-50 YO of the total genome. These sequences also show a low intraspecific variation of the monomeric se- quence, are A + T rich, and have frequent clusters of A or T 2 3 residues. Potential secondary and/or tertiary structures and a homogeneous distribution among all the chromosomes are also observed (WEITH 1985; PETITPIERRE et al. 1988; DAVIS and WYATT 1989; UGARKOVIC et al. 1989, 1992; JUAN et al. 1991, 1993; PLOHL et al. 1990, 1993).

Misolampus goudoti Er. (Tenebrionidae, Cole- optera) is a darkling beetle species with 2n=20 metacentric/submetacentric chromosomes present- ing large centromeric and some small telomeric C-positive bands. These bands constitute about 58 YO of the total chromosome complement in mitotic metaphases (JUAN and PETITPIERRE 1989). Here, we have characterized and cloned two different satellite DNA families of Misolam- pus goudoti to compare the relationships and characteristics of these highly repetitive sequences with those of other tenebrionids, in the hope that these data may shed some light on the evolu- tion and possible significance of these DNA se- quences in darkling beetles. Furthermore, the digestion of fixed chromosomes with some of the restriction enzymes used on naked DNA, can give information on the chromosomal orga- nization and localization of these satellite DNA sequences.

180 J. PONS ET AL. Hereditas 119 (1993)

Material and methods DNA extraction and digestion

Misolampus goudoti was collected in the field from several localities in Mallorca Island (Spain). For the DNA extraction, pooled adults were dissected and the digestive tube discarded, the remaining soft tissues used for homogenization. A standard method of DNA extraction described elsewhere (JUAN et al. 1993) was followed for obtaining genomic DNA from the beetles. The restriction enzymes used in this study were purchased from Boehringer-Mannheim and DNA digestions were performed at standard conditions ( SAMBROOK et al. 1989). DNA digests were run on 1.5-2 % agarose gels made in TBE (SAMBROOK et al. 1989) with ethidium bromide. In some cases the photo- graphs obtained from the gels were scanned by means of a densitometer.

Cloning and labelling of probes

Three different genomic DNA bands from Miso- lampus goudoti were used for cloning. One of about 200 bp, obtained by cleavage with EcoRI, was cloned in the EcoRI site on M13 mp18, and an- other of about 250 bp, obtained by cutting the DNA with ClaI, was inserted in the AccI site of the same vector. Finally, a band of about 1200 bp, obtained by cleavage of genomic DNA with PstI, was cloned in pUC18 plasmid. The potentially recombinant plaques or colonies obtained were tested for the presence of the DNA specific bands using a portion of the eluted genomic bands taken for cloning and labelled with digoxigenin-dUTP as probes in Southern hybridizations with the replica- tive forms of recombinant phages or the recombi- nant plasmids purified by standard protocols (SAMBROOK et al. 1989). We called the three different probes pMg200, pMg250, and pMg1200 for the DNA inserts obtained with EcoRI, ClaI, and PstI restriction enzymes, respectively. Probes were labelled with digoxigenin-dUTP, by the ran- dom priming method (FEINBERG and VOGEL- STEIN 1983).

Southern hybridizations

We used the capillary method for DNA transfer to nylon Hybond-N membranes (Amersham) and ex- posure to ultraviolet light for cross-linking the DNA to the filter. Hybridization conditions were essentially those recommended by Boehringer- Mannheim for the “DNA labeling and detection kit nonradioactive”. The immunological detection of the hapten digoxigenin with anti-digoxigenin conjugate to alkaline phosphatase was followed by either a color or a chemiluminescence reaction.

Chromosome preparations and banding

Chromosome preparations were obtained from male gonads by the methods described previously (JUAN et al. 1991). C-banding and fluorochrome staining were made according to the methods re- ported by SUMNER (1972) and SCHWEIZER (1976) respectively. The procedure of in situ digestion with restriction enzymes was described elsewhere (JUAN et al. 1991).

Results Detection of satellite DNAs of M. goudoti

Genomic DNA from Misolampus goudoti was cleaved with several restriction enzymes. Some of them gave conspicuous bands corresponding to repetitive DNAs. A prominent genomic DNA band of about 200 bp obtained by digestion with EcoRI was cloned in M13. One of the obtained recombinant clones, the pMg200-6, was used as probe in a Southern experiment where M. goudoti genomic DNA was digested with seven restriction enzymes. A complex hybridization pattern showing bands between - 150 and - 1000 bp was present when the genomic DNA was digested with EcoRI, HinfI, and AluI and hybridized with pMg200-6 (not shown). To investigate more deeply this hy- bridization profile, partial digestions of M . goudoti genomic DNA were undertaken with EcoRI. A series of bands arranged in a ladder pattern ap- peared in this case, with an increasing fluorescence intensity of an ethidium bromide-stained DNA band of about 200 bp when the digestion time was prolonged (Fig. la). Multimers up to the octamer or even more were visible in shorter digestions, showing a tandem arrangement of this sequence in the genome. Also, a clear band of about 270 bp

Sequencing

Single stranded DNA obtained from the MI3 re- combinant clones was used for sequencing by the Sanger dideoxy methods (SANGER et al. 1977) using Sequenase or T7 polymerase.

Heredifas 119 (1993)

1 2 3 4

SATELLITE DNA OF MISOLAMPUS GOUDOTI 181

700 4 600

, 1200

700

500

,270

4 200 * 200

Fig. la-c. Agarose gel electrophoresis of genomic DNA from M . goudoti partially digested with EcoRI (a). The DNA was digested with the enzyme at a concentration of 1 U/bg DNA during 15 min (lane 2), 30 min (lane 3) and 1 h (lane 4). In lane I was run a partial digestion of T. moliror DNA, giving a ladder of multiples of 142 bp as DNA marker. This gel was Southern transferred to a filter and hybridized with pMg200-6 labelled probe (b). After washing the signal, the same filter was hybridized with pMg1200- 2 labelled probe (c). The approximate size of a series of bands in a. b and c is indicated at margin in base pairs.

and multiples thereof were visible in the partial digestions, resulting in two ladders of bands with a phase difference of about 70 bp. Furthermore, nu- merous intermediate bands with fainter intensity were also visible in the EcoRI partial digestions. This gel was transferred to a nylon membrane and hybridized with pMg200-6, showing a clear series of hybridizing bands of 200 bp and multimers up to the octamer, and intermediate fainter bands, including the 270 bp and multimers (Fig. lb). This suggests that frequent events of unequal crossing- over within the 200 bp unit are rendering different size-variants of the main repeating unit. A promi- nent DNA band of about 700 bp produced by a prolonged digestion with EcoRI did not show cross-hybridization with pMg200-6, suggesting the presence of more than one repeated DNA class.

Another genomic DNA band of about 250 bp was obtained by digestion of M . goudoti DNA with ClaI and subsequently cloned. The recombi- nant clone pMg250-1 used as probe in Southern hybridizations with genomic DNA digested with several restriction enzymes, including EcoRI, gave a pattern different from that obtained with

pMg200-6 (Fig. lc), confirming the presence of more than one family of highly repetitive DNAs in M . goudoti. A unique band of approximately 1.2 kb, obtained with PstI, plus bands of smaller sizes with other restriction enzymes showed cross- hybridization with pMg250-1. The tandem repeti- tion of this DNA band was supported by partial digestions of genomic DNA with PstI (Fig. 2a-b), though in this case the hybridization signal was present in the monomeric band of 1.2 kb and in multimers up to the tetramer, suggesting that the satellite is in clusters of at least 4.8 kb in the genome of M . goudoti.

Photographs of gels obtained by digestions of DNA using the enzymes cited above were scanned with a scanning densitometer for quantifying both satellites of the M . goudoti genome. The peaks obtained in separate scans of different digests were estimated as 7 YO and 25 % for the EcoRI and PstI bands, respectively. Since this refers to the monomeric bands only, and dimers are also appar- ent, these percentages represent a minimum esti- mate. Assuming a nuclear haploid genome of about 3.29 x lo8 bp in M . goudoti (JUAN and PE-

Herediias 119 (1993) 182 J. PONS ET AL.

1 2 the mutational events preferentially occumng in the flanking regions of the 61 bp motif.

Two clones of the 250bp band obtained by digestion of genomic DNA with Clal were se- quenced showing differences in length; pMg250-1 has an insert 259 bp long while pMg250-2 was 253 bp. The sequence is A-T rich (58.7 YO, average of the two) with many clusters of A or T rich motifs (Fig. 3b).

In situ digestion with restriction enzymes

Misolampus goudoti has a diploid number of Fig. 2a and b. Partial digestion with PstI of genomic 2n = 20 chromosomes; 9 pairs of meta or submeta- DNA from M. goudoti (a, lane 1). The size marker was centric autosomes, a metacentnc X-chromosome, lambda DNA digested with Hind111 (a, lane 2). The gel and a dot-like Y-chromosome. Large pericen- was transferred to a filter and hybridized with pMg1200-2 tromeric C-bands are visible in all chromosomes labeled probe (b). The approximate size of three DNA and small telocentric bands, in some of them

(JUAN and PETITPIERRE 1989). Staining with bands is indicated at margin in base pairs.

distamycin/diamidinophenylindol ( DA/DAPI), a

TITPIERRE 1989), gives a minimum figure of about 120,000 copies for the EcoRl and circa 70,000 copies per haploid genome for the PstI family of highly repetitive sequences in M. goudoti.

Six species of the genus Tribolium plus Tenebrio molitor were tested for the presence of M. goudoti satellite-like sequences. Southern experiments indi- cated an absence of both M, goudoti DNA se- quences in these related species of the same family. Homology comparisons between the M . goudoti satellite sequences to the published major satellites of other tenebrionids like those of Tenebrio moli- tor, Tribolium freemani, and T. confusum did not give significant similarities either.

DNA sequence analysis

Sequence analysis of 7 different clones of the pMg2OO monomeric repeat gave a consensus se- quence of 196 bp with unique recognition sites for EcoRI, Hinfl and Sau3AI (Fig. 3a). The sequence is 64.3 YO A-T rich and has runs of A or T 2 3 nucleotides spread over its length. A total of 26 independent substitutions and one insertion was detected when comparing each sequence with the consensus. There is an average of 3.7 substitutions per monomer, which corresponds to a sequence variation of about 1.9 YO. The sequence also shows two internal direct repeats of 61 bp in positions 41 to 101 and 114 to 174, which share about 88.5 YO similarity with each other. These direct repeats have, in turn, a perfectly repeated core of 31 bp,

fluorochrome which binds preferentially to A-T rich chromosomal regions, showed few or even null longitudinal differentiation across the chromo- somes. However, the preferentially G-C rich bind- ing chromomycin A,(CMA,) produced a faint fluorescence in centromeric positions compared to the one observed in the distal parts of chromoso- mal arms (not shown).

Some of the restriction endonucleases which in- duced genomic bands in naked DNA in M . goudoti were used in in situ digestions. EcoRI extracted DNA from centromeric positions of all chromo- somes, resulting in the formation of gaps in those regions (Fig. 4a) and completely abolishing the staining of centromeric heterochromatic blocks in pachytene. On the other hand, PstI digested cen- tromeric regions of specific chromosomes only, like pair no. 1 (Fig. 4b). Finally, Hinff induces an unstained gap in an interstitial region of the X- chromosome (Fig. 4c).

Discussion In this study two satellite DNA families from the beetle Misolampus goudoti have been analyzed and characterized. The EcoRI family shows character- istics of a DNA sequence with extensive tandem repetitions in the genome. The length of the main monomeric unit is very close to those present in other tenebrionids like Tenebrio molitor, Triboliwn freemani, Palorus ratzeburgii, T. confwum, and T. anaphe which are 142, 166, 142, 158 and 170 bp,

Heredim 119 (1993) SATELLITE DNA OF MISOLAMPUS GOUDOTI 183

a I 10 I 20 I 30 I 40 I 50 I 60 1 GAATTCTATG TGCGTGTACC CTTAAACCAC GATTCTGCAT ctagaaagca ccgttctcat 60

61 gtaaaaacct acattaacac agatttggag taagtttgag aTCAATTCTG TAActagaaa 120 121 gcacattcct catgtaaaaa cctacattaa cacagatttc tagtaagtct gagaCCTAAG 180 181 AAATGCCTAA ACTTAT 196

b I 10 I 20 I 30 I 40 I 50 I 60 1 GTCGATTGGA ACCAAAATCG AGCCGATAGC TCGAGTTTTG AGCGAGTAAT CGGCGACTGA 60

61 AGTTCAAAAT TTAGAAAAAA ATTTGAAAAT GCCTTTATCG CGCTCTGCAG CGCAAACGAA 120 121 GCTCCGAATC GAACTTTTGA GGGCAGATTC TGAAAGAGCG GATCAAGATA CATAAAGTTC 180 181 ACCCAATTCT TTGTGGATAT GGCGCGAAAT GAAGAAGAGC CTACAGAAGC GGATTTTTAA 240 241 AAAATTGACA AATTTTTCA 259 Fig. 3a and b. Nucleotide consensus sequence of M. goudoti satellite DNA monometric unit (a), the beginning and the end are delimited by the restriction site EcoRI. Note the presence of two internal direct repeats indicated in small letters. Nucleotide sequence of the probe pMg250-1 (b).

Fig. 4a-c. Mitotic spermatogonial chromosomes of M . goudoti after digestion with &OM (a); note the low staining of centromeric areas (the chromosome pair 1, in which this is clearer, is marked by arrowheads). Digestion of a mitotic metaphase (incomplete) with PstI (b); chromosome one shows decrease of staining at centromere (arrowhead). Digestion with Hinfl (c), the X-chromosome shows an interstitial gap (arrowheaded). Bar represents 5 bm,

respectively (PETITPIERRE et al. 1988; UGARKOVIC Tribolium freemani as demonstrated by in situ hy- et al. 1992; JUAN et al. 1993; PLOHL et al. 1993). In bridization and/or in situ digestions (DAVIS and all these cases the repeating units are organized in WYATT 1989; JUAN et al. 1991, 1993). In addition, large tandem arrays and are localized at cen- these sequences have in common a high A + T tromeric positions, at least in Tenebrio molitor and content, ranging from 58.5 YO to 73 YO. In the

184 J. PONS ET AL. Herediras 119 (1993)

present case, the EcoRI satellite of Misolampus goudoti has a high but intermediate range of A + T content of 64.3 % and a length of 196 bp. The average sequence variation encountered in 7 differ- ent monomers is similar to that found in the satellites of Tenebrio molitor and Tribolium con- fusum, which are about 2 % (UGARKOVIC et al. 1989; PLOHL et al. 1993). From the sequence anal- ysis of 7 clones (1372 nts) we can conclude that there is a similar tendency of G, C and A, T to the substitution, but transversions are in excess with respect to transitions (transversions/transitions =4.2). Two internal direct repeats of 61 bp are probably related with the ancestral DNA sequence which originated the present 196 bp monomer by amplification and/or unequal crossing-over mecha- nisms (JOHN and MIKLOS 1988). On the other hand, the origin of the monomeric minor repeating unit of 270 bp can be explained by unequal cross- ing-over within the main 200 bp repeats in the regions of internal direct repetition. This unequal crossing-over preferentially produces new size units of about 2/3 and 1 + 1/3 ( - 122 and -270 bp), although other intermediate units might also be produced with less probability by this recombina- tional process.

The sequence has also short motifs rich in A and T nucleotides that could be significant in making potential secondary structures. RADIC et al. (1987) and MARTINEZ-BALBAS et al. (1990) have shown that a large range of satellite DNA sequences show numerous clusters of (A-T) 2 3 residues and that this can be related with DNA curvature as demonstrated by retarded migration of the repeat in non-denaturing polyacrylamide gels in mouse, rat and alfa-monkey satellites. Se- quence-induced curvature has been demonstrated in the Tenebrio molitor and Palorus ratzeburgii satellite repeats as well, and models consisting in similar solenoid structures with left-handed super- helix have been proposed for these satellite DNAs (PLOHL et al. 1990; UGARKOVIC et al. 1992). The implications of DNA curvature in satellite DNAs are not well established but it has been suggested that they can be related with chromatin organiza- tion and condensation by nucleosome phasing (MARTINEZ-BALBAS et al. 1990).

Another satellite DNA family (PstI family) in Misolampus goudoti with a higher monomeric length has also been partially characterized. This repetitive DNA has a monomer of 1.2 kb arranged in the genome in tandems of at least four of these units, as shown by partial digestions with PstI.

In situ digestion with restriction enzymes is a method able to localize DNA sequences in chro- mosomes, especially those sequences which are re- peated in the genome (for a revision, see LOPEZ-FERNANDEZ et al. 1991). In situ digestions with the same restriction enzymes used to charac- terize the satellite DNA sequences in M . goudoti show some interesting results. HinfI and EcoRI, enzymes which cut the EcoRI satellite family in monomers of 196 bp and multiples (although PstI family is somewhat digested by both enzymes), induce different banding patterns when used in situ. EcoRI extracts DNA in pericentromeric posi- tions of each chromosome while HinfI does it preferentially in an interstitial region of the short arm of the X-chromosome, giving in each case a decrease of Giemsa staining. The fact that the two enzymes differ in the produced chromosomal band- ing while the effect on naked DNA is very similar, points to a differential accessibility of either en- zymes to the centromeric heterochromatin. EcoRI cuts and extracts DNA from the EcoRI-satellite in fixed chromosomes, therefore showing a centro- meric localization of these sequences. The pattern obtained with EcoRI resembles the one obtained by CMA, staining in mitotic chromosomes, i.e., a faint fluorescence in the pericentromeric regions of all chromosomes, in accordance with the rela- tively low G + C content of the EcoRI family of satellite DNA.

PstI used for in situ digestion gave gaps in centromeric regions of some chromosomes like in pair no. 1. It has been suggested that large DNA fragments are unable or much more difficult to be extracted from fixed chromosomes by in situ diges- tions than are short fragments (MILLER et al. 1983). If this is true, there would be expected a lesser in situ DNA extraction effect by PstI than by EcoRI, since the minimum lengths of rendered satellite DNA fragments are 1.2 kb and 196 bp in conventional digestions of naked DNA, respec- tively. Nevertheless, centromeric C-bands are larger in M . goudoti chromosomes than the gaps caused by either EcoRI, PstI, or HinfI. Further- more, any of these three endonucleases seems to be able to differentiate the telomeric heterochromatin, visible as small C-positive bands. In situ hybridiza- tion experiments are currently being performed to solve these questions.

Acknowledgements. -We are indebted to Prof. G. M. Hewitt (UEA, Norwich) for laboratory facilities. The help of Mrs. Inger Arnau in DNA sequencing and that of Dr. M. J. Arranz in

Hereditas 119 (1993) SATELLITE DNA OF MISOLAMPUS GOUDOTI 185

revising the English style of the manuscript are also acknowl- edged. The suggestions of two anonymous referees substantially improved the manuscript. This work has been supported by project DGICYT number PB90/0357 (Spain) and “Acciones In- tegradas Hispano-Britanicas” number 8A.

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