Volume 11 Number 20 1983
`
`Nucleic Acids Research
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`Structure and function of an AT—rich, interspersed repetitive sequence from Chironomus thummi:
`solenoidal DNA, 142 hp paltndrome-frame and homologies with the sequence for site-specific
`recombination of bacteria] tmnsposons
`'
`
`Norbert Israelewski‘
`
`Ruhr—UnivcrsitaI Bochum, MA 5, Lehrstuhl fflr Genctik, D-44630 Bochum, FRG
`
`Received 19 July 1983; Revised and Accepted 3 October 1983
`
`ABSTRACT
`
`thummi contains a repetitive AT—rich 118 bp sequence
`Chironomus thummi
`mainly in the centromere regions and elsewhere in the genome (1). A large
`cluster of repeats is regularly present in the non-transcribed spacer of
`rDNA. Dimers and multimers of the repeat migrate slower in small pore gels
`than would be expected from their size. The results indicate a solenoidal
`structure with a coil girth of appr. 350 bp. This structure is most probably
`due to a highly periodic positioning of di-nucleotides of the type purine -
`purine or pyrimidine-pyrimidine with distances of appr. 10 bases.
`In a clus-
`ter pf 118 bp repeats, regions of dyad—synnmtry are positioned such that a
`142 - 2 bp palindrome—frame is generated. Evidence is presented favouring the
`assumption that the repeat functions primarily in sister chromatid exchange.
`
`INTRODUCTION
`
`Among Chironomides the two subspecies Chironomus thumni
`
`thummi and Eni-
`
`interest since they represent a
`ronomus thumi piger have received special
`system which allows the first steps in species separation to be studied. This
`
`separation process is accompanied by a geometric increase of DNA in polytene
`
`chromosome bands mainly at the centromeres and neighbouring regions of the
`
`increase of DNA is in some
`thumi genome (2, 3). The local
`Cironomus thumi
`way coupled with an increase in the amount of repetitive DNA sequences (4, 5)
`
`which is also visible in an increase of the amount of C - banding DNA (6). A
`
`comparable situation is found in Drosophila sibling species where it has been
`emphasized that apparent differences in the amount of highly repetitive DNA
`
`sequences accompanies species separation (7, 8). The variation in the highly
`repetitive portion of the genome might also include regions carrying infor-
`
`mation for proteins (9).
`In the two Ch.thunmi subspecies a centromeric cluster of an AT-rich re-
`
`petitive 118 bp sequence has been characterized (1). This cluster has been
`
`magnified and dislocated in §h;_th; thummi and is also found at those sites
`where the DNA content in bands has increased (1). The fact that this sequence
`
`
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`© IRL Press Limited, Oxford, England.
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`Nucleic Acids Research
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`has also entered the non-transcribed spacer of rDNA led us assume that the
`
`repetitive sequence may function in promting sister chromatid exchange (SCE)
`
`(10). A high frequency of SCE would provide the redundant gene cluster with a
`
`greater evolutionary flexibility (11). Evidence has been accumulated favour-
`
`ing this hypothesis (12, 13 and this report).
`
`MATERIAL AND METHODS
`1. DNA
`
`Cloned rDNA of §h;_th thummi pCtt 1505 and pCtt 1507 was kindly provided
`by E. R. Schmidt (17). The plasmid DNA was further purified from contaminating
`
`RNA by ethanol precipitation in the presence of 2.5 M ammonium acetate. If
`desired,
`the rDNA insert was separated from the vector pBR 328 by EcoRI re-
`
`striction of the clone followed by preparative gel electrophoresis and
`electroelution of the DNA.
`
`2. DNA Restriction and Gel Electrophoresis
`Enzyme incubations were perfonmed as described by the producer (Boehrin-
`ger). Restriction fragments were separated on 3 m horizontal slab gels in a
`
`tris - phosphate — EDTA buffer system (14). Agarose gels were prepared accord-
`ing to (15, 16). Polyacrylamide gels (
`1
`: 20 ratio N,N'methylenbisacryl-
`
`amide : acrylamide) were reinforced with 0.5% agarose. QX174 - HaeIII and
`A- HindIII
`fragments were used as size markers and the gels analysed by ethi-
`
`dium bromide staining. Negative prints are shown.
`
`RESULTS
`
`The restriction map of the cloned rDNA of £h;_th; thunmi pCtt 1507 is
`shown in figure 1. This clone contains appr. 22 x 118 bp repeats (ClaI re-
`
`peats) which has been sequenced (17). The map is identical with that of pCtt
`1505 except that clone pCtt 1507 exhibits stability in the number of ClaI
`
`repeats after replication in E; 5911 unlike pCtt 1505 which is highly un-
`stable in the number of Clal repeats (17). Sequence data have shown that the
`
`stable clone 1507 contains a base substitution at the right end of the Clal
`
`repeat cluster (18) and several uncharacterized mutations in the vector pBR
`
`repeats into the vector (Israelewski, unpubl)
`328 including the spread of Clal
`It was
`therefore decided to separate the rDNA insert from the vector by EcoRI
`restriction and preparative gel electrophoresis.
`
`Measurements of the length of the EcoRI fragment based on its mbility
`
`on gel electrophoresis gives rise to different values which are apparently
`
`dependent on the pore size of the gel. Higher percentage gels slow down dis-
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`Nucleic Acids Research
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`Ecokl Hmdlfl
`
`Smal
`Clal
`
`q
`
`CIA!
`
`Clal
`
`Hmdlll
`Ecol?!
`
`Hulfl Hnlil
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`Hull! Hull! Hull! I-tpall
`
`Hpall
`
`E:
`pC!(I507
`.
`rug
`
`rDNA cistron pCtt 1507
`thummi
`th.
`Restriction map of a cloned Ch.
`1
`.
`Fi
`according to 10. 17). Of
`the HpaII sites only the two in the NTS are shown.
`The asterix indicates a Clal site that is present in most of the genomic rDNA
`cistrons, but is absent
`in cloned rDNA.
`
`proportionately the migration of the fragment.
`
`In 0.5%, 1.0%, 1.5% and 2.0%
`
`agarose gels lengths of 11.0 kb, 12.9 kb, 15.5 kb and 40 kb were measured
`
`respectively. when HaeIII restricted DNA is electrophoresed it is only the
`NTS carrying fragment which migrates anomalously. The same is true for HpaII
`
`restriction fragments. (fig. 2A). After ClaI digestion it is seen that the two
`
`flanking segments of the ClaI repeat cluster migrate correctly in 2.0% agarose
`
`gels. Comparisons of the gel dependent shift in the sizes of the EcoRI, HaeIII
`
`and HpaII fragments indicate that the shift is more pronounced if the ClaI
`
`repeat cluster is flanked by large DNA segments which alone do not show the
`
`shift (fig. 2B).
`
`In the case of the HpaII fragment there additionally appears
`
`a faint band without shift (arrows, fig 2A, clearly seen only in 1.5% and
`
`2.0% agarose gels). Since no HpaII fragment greater than 2.6 kb should exist
`
`besides the NTS—HpaII-fragment (3.7 kb) it is possible that in few plasmids
`
`one HpaII site is modified (at the left of 28S) resulting in the 3.7 kb band.
`
`For analysis of the gel-type dependent shift in the sizes of monomers
`
`and multimers of the ClaI repeat the clone pCtt 1507 was digested incompletely
`
`with Clal and the DNA was run on polyacrylamide (PAA) gels. The shift is
`measured as percentage deviation from the real value. Three typical curves
`
`the mono-
`three PAA gels (2.0% - 2.5% - 3.0%)
`In all
`are shown in figure 3A.
`mr size was measured to be 116 bp corresponding to a deviation of -2% rela-
`
`tive to the 118 bp fragment of ¢X174-HaeIII. with 2.0% gels the shift of di-
`mers and multimers increases up to +20% for six ClaI repeats. Further increas-
`ing oithe number of the repeats does not contribute to a further increase of
`
`the shift. Hith 2.5% PAA gels the curve shows systematic steps resulting in
`
`kinks of the curve at intervals corresponding appr.
`
`to multiples of three ClaI
`
`repeats. Hith 3.0% PAA gels the shift increases dramatically up to nine re-
`
`peats; further multimers cannot be measured in this type of gel.
`In each case care was taken to avoid partial denaturation of the DNA
`
`which alters its mobility on electrophoresis. If the gel heated up due to a
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`Nucleic Acids Research
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`I K
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`2I
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`(kb)
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`high current,
`
`the curves shown above are not exactly reproducible. However,
`
`partial denaturing of the Clal repeat DNA under controlled conditions (
`
`2M
`
`leads to a systematic enlargement of
`urea in the sample application buffer )
`the steps in the curve for 2.5% PAA gels, especially at higher numbers of
`
`ClaI repeats (fig. 3B).
`It has been noticed that clone pCtt 1505 is not stable during replication
`The
`
`in E; coli
`leading to a decrease in the number of ClaI repeats (17).
`presented data of asecondary structure of the ClaI repeat and the possible
`
`interference of a recombination system of §;_coli with the Clal repeat se-
`quence (17 and discussion) prompted us to re-evaluate the elimination process
`
`of Clal repeats in §;_goli. Using a single colony isolate for the DNA prepa-
`ration,
`the restriction with HaeIII results in a ladder of NTS fragments with
`
`size intervals of 120 bp (fig. 4A). It is interesting that certain fragment
`
`In the lower
`sizes are prefered during the elimination process in E; coli.
`molecular weight region it is seen that these fragments have distances of
`
`is cultivated succes-
`3 x 120 bp or multiples of that (arrows). If E; coli
`sively four times overnight,
`three prominent HaeIII fragments accumulate
`
`which have also size intervals of 360 bp (3 x ClaI repeat, fig. 4B). Thus,
`it is suggested that recombination in the cloned rDNA occurs at a defined
`
`site in the Clal sequences modulated by the structural feature of the DNA.
`
`DISCUSSION
`
`Structure and function are two aspects of living matter (19). Consider-
`
`ing the results suggesting a defined secondary structure of the ClaI repeat
`
`DNA of gn; tn; thumni it may be anticipated that one can also find a function.
`Thus, sequence data (17) were analysed.
`
`I.
`
`10 bases periodicity and bent helical DNA structure
`Trifonov (20) has predicted that the DNA axis is curved if some di-
`
`nucleotides of the type purine-purine or pyrimidine-pyrimidine have the ten-
`
`In a long DNA fragment
`dency to be repeated with a period of about 10 bases.
`this would lead to a solenoidal DNA structure which is stable in a DNA mole-
`
`cule free of protein.
`
`In the ClaI repeat sequence an almost perfect 10 bases
`
`A) Agarose gel electrophoresis of the rDNA insert of pCtt 1507 after
`2
`.
`Fi
`restriction with the enzymes indicated at the top of each lane. The NTS con-
`taining fragments (-) migrate anomalously in 1.0% - 2.0% agarose gels. On eli-
`mination of the ClaI repeat DNA by digestion with ClaI
`the two flanking frag-
`ments (4) of the repeat cluster migrate correctly in 2.0% agarose gels. Arrow:
`see text. B) Plot of the measured size of the NTS containing fragments as a
`function of the percentage of agarose.
`
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`6989
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`Nucleic ACldS Research
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`:\:‘‘O
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`
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`in the apparent sizes of monomers and
`Fig. 3 Gel-type dependent shift (1)
`mu
`imers of the Clal repeat on electrophoresis in 2.0% - 3.0% polyacrylamide
`gels. The shift is plotted against the number of Clal repeats. The fragment at
`0.34 kb (o)
`is derived from 28S rDNA and it does not show the shift. Sample
`application buffer contained A)
`1 M urea, B) 2 M urea.
`
`In accordance with Trifonov's findings is
`periodicity is visible (fig. 5).
`the fact that purine-purine dinucleotides have a maximum when the pyrimidine
`
`-pyrimidine ones have a minimum. Marini et al.
`
`(20) have shown that the se-
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`Nucleic Acids Research
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`-&
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`Elimination of ClaI repeats during
`. 4
`Fi
`rep ication in E. coli. The ladder of HaeIII
`fragments of cloned rDNA pCtt 1505 is shown.
`These fragents contain the NTS region.
`0.8% garose gel. Arrows: see text. A) On
`cultivation of E. coli overnight, B) On four
`successive overnight-cultivations of E; coli.
`
`too slow migra-
`quence dependent curving of DNA of the type above results in a
`tion of the DNA structure in small pore gels. Here it is demonstrated that
`
`flanking ‘linear’ DNA segments enhance the anoalous migration. Furthermore,
`
`an investigation of the migration of monomer and multimers of the ClaI repeat
`
`a stepwise altering of the mobility of the DNA is observed every 350 bp (appr.
`3 x repeat length). It is likely that this periodicity reflects the coil girth
`of the DNA under consideration, since it is this parameter of a spiral which
`
`is coupled with periodic repetitions of a certain DNA length.
`
`The observed periodic alteration in the mobility of the Clal repeat DNA
`
`structure may be explained statistically in that each coil contributes to
`
`align the DNA structure at a pore of the gel. If so,
`
`the actual pore size
`
`of a gel determines the degree of alignment which is necessary for the DNA
`
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`Nucleic Acids Research
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`. 5 Distribution of the dinucleotides of the type A) purine~pyrimidine.
`Fi
`B) purine-purine, C) pyrimidine—pyrimidine. Frequencies of the occurrence of
`the dinucleotides were recorded within distances of three bases along the 118
`bp sequence.
`
`structure to snake through the pore. Once aligned,further coils passing the
`
`pore do not significantly slow down the migration of the DNA structure (fig.3)
`
`II.
`
`142 bp distances between regions of dyad-sflyyetry in the clustered repeat
`It has been suggested that the sequence-dependent curving of the DNA axis
`
`In this context it seems noteworthy
`facilitates its folding in chromatin (22).
`that other potential structural elements. regions of dyad-symmetry, are ob-
`
`viously coupled with a systematic positioning message present
`
`in the Clal
`
`repeat DNA. It is shown in figure 6 that in a cluster of seven Clal repeats
`
`palindromes are positioned such that a 142 1 Zbp frame is generated,
`
`this
`
`142 bp
`
`142 bp
`
`
`
`
`144 bp
`
`140 bp
`
`Regions of dyad-symmetry over at least 5 bp without mismatch psi-
`. 6
`Fi
`tionea in a cluster of seven ClaI repeats (schematically delimited by bars).
`Numbering of the bases refers
`to Schmidt et al.
`(17). Only those palindromes
`are shown which are positioned in the 142 1 2 bp fram (circled bases).
`
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`Nucleic Acids Research
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`2211-A
`A- 1
`1- A
`A- 1
`9- c
`1- A
`A -1
`G- c
`
`1- A
`
`142 bp
`
`134A-T
`A- 1
`1- A
`A- 1
`1- A
`1- A
`1 -A
`
`<\?:::)
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`T-A104
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`1 -A
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`A -1
`T —A
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`1 -A
`A -1
`G -c
`
`1 -A
`
`142 bp
`
`142 bp
`
`Regions of dyad-symmetry over two 234 bp Alul repeats of Droso hila
`7
`.
`Fi
`me anogaster (schematically delimited by bars). Numbering of the bases refers
`to Miller et al.
`(24). Only those palindromes are shown which are positioned
`in the 142 bp frame (circled bases).
`
`being in excellent agreement with the core DNA length in a nucleosome. The
`
`DNA looped out by the palindromes is not constant in length but has,in most
`cases,
`lengths of appr. 30 bp. It is evident that such a frame cannot evolve
`
`by chance and we have no explanation other than the suggestion that nucleo-
`somes select their binding sites by utilizing these structural features.The
`
`positions of the 142 bp-frame palindromes in the solenoid appears to be not
`
`entirely random: appr. each half
`
`turn of a coil wuld be the potential site
`
`for palindromic refolding. The palindroes shown in figure 6 represent one
`third of the number of palindromic sequences present in the Clal repeat. A
`
`detailed description of all palindromic sequences together with the statisti-
`
`cal analysis is in preparation. Briefly, a systematic arrangement is found
`
`for all 15 palindromes utilising the basic frame of 142 i 2 bp.
`
`In the african green monkey 172 bp repeat which is known to be phased
`
`with nucleosomes (23, 49)
`
`the perfect palindromic sequence GATATTT - 31bp —
`
`AAATATC is capable of looping out
`
`the presumed linker DNA region thus genera-
`
`ting a 141 bp frame in the clustered repeat. Recently the positions of the
`
`phased nucleosoes on the 172 bp repeat have been determined (49). The nucle-
`osome core bounderies of the most frequent frame F fit the above mentioned
`
`palindrome with an accuracy of 3 bp.
`
`Another example of systematically arranged palindromes is present in the
`
`234 bp repeat of the rDNA of Drosophila melanogaster. These repeats serve as
`‘loading sites‘ for RNA polymerase I and they can augment
`the transcription
`
`of the rDNA unit (24, 25, 26). This frame, however,
`is somewhat differently
`organized when compared to the one in §h;_th; thummi (fig. 7).
`
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`AAA/«G6:
`:9 T
`A
`-AAA? cc
`GA"jTA[%
`thumm
`m _ ll.llll§hcAlIlc.llcil.ll.’:c§ 'Tl...li.llllGG1ll illlll
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`[tie
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`l‘llIéliillllllmflfififiliagllléglllmli i if
`
`-10
`
`I
`
`Sequence homologies between the 118 bp ClaI repeat (numbering of the
`Fig. 8
`Bases refers to Schmidt et al.
`(17) and the sequence for site-specific re-
`cobination (resolution) of the bacteria] transposon TN3 (Grindley et al
`,27).
`Underlined are the three binding sites for the bacterial resolvase enzyme. The
`actual site of recombination in TN3 is indicated by the arrow. Homology is 64%.
`
`these examples lead one to believe that palindromes in the
`In conclusion,
`142 bp frame are important
`infonnational structures for assembly and / or
`
`the palindrome frame would
`In addition,
`function of repeated DNA sequences.
`not allow that a random variation in the repeat size can be accepted.
`
`Homologies with the sequence for site-specific recombination of
`III.
`bacterial
`transposons of the type TN3
`has 64% homology
`It appears highly surprising that the ClaI sequence
`with the bacterial sequence which is known to mediate site-specific recombi-
`
`nation of transposonsxfl and TN3 during DNA replication in E; coli (fig. 8).
`But with this fact in mind four phenomena coupled with the presence of Clal
`
`repeats of Eh. th; thummi become explainable: 1. The Clal repeat is found at
`sites where a geometric increase of DNA segments have occured (1, 3),
`2. An extensive variation in the number of ClaI repeats is found in the NTS
`
`regions of rDNA cistrons (10). 3.
`
`It is difficult to clone the rDNA of Eh; th;
`
`thummi
`
`in E; coli. One of the two clones obtained is highly unstable in the
`
`(17 and results). The
`number of ClaI repeats during replication in E; coli
`second clone is stable, but this is coupled with several mutations (see re-
`
`In certain crosses of Eh; th; thummi x Eh; th; 3133: a high inci-
`sults). 4.
`dence of chromsomal mutations is observed at sites with high number of Clal
`
`repeats (28).
`Comnon to all
`
`these phenoena is that some fonm of exchange is necessary.
`
`Phenomena 1
`
`- 3 involve unequal exchange and phenomenon 4 illegitimate ex-
`
`there is growing evidence that the ClaI repeat together
`In conclusion,
`change.
`with its structure serves a role in the generation of sister chromatid ex-
`
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`change (SCE) which might be unequal. It is known that SCEs are produced during
`
`DNA replication (29,30,31). Unequal SCE is especially favourable for redundant
`genes in order to ensure the horizontal evolution within the gene cluster (11,
`
`the ClaI repeat DNA and possibly other related AT-rich se-
`32). Additionally,
`quences canpunctuate genomic DNA so that the exchange event is necessarily
`
`confined to the repeats. This corresponds to the conception for meiotic ex-
`change that chromosomal structures have arisen in a functional connection with
`
`the gene regions are pro-
`exchange events in lampbrush chromosomes. Thereby,
`As a matter of fact, punctuation of the
`
`tected from disintegration (34,35,36).
`
`genomic DNA by AT-rich sequences is characteristic of eukaryotes (37,38).
`
`Finally, it should be emphazised that the present investigation adds evi-
`dence that repetitive DNA sequences can be involved in recombination systems
`
`(39 - 44).
`
`In plant species a family of conserved middle repetitive DNA se-
`
`- 2 kb plays an important role in meiotic re-
`
`lengths of 1
`quences of modal
`combination (45,46,47)
`
`A similar sized DNA sequence (1.1 kb)
`
`is apparently
`
`involved in the meiotic exchange process of the Diptera Phryne cincta (Isra-
`elewski,
`in prep.). Furthermore, it appears that at least some of the re-
`
`combination mechanisms have been conserved essentially before pro- and
`
`eukaryotes diverged. This can also be deduced from the fact that a repetitive
`
`DNA sequence from the kangeroo rat Dipodomys ordii
`
`is recognized by the re-
`
`In this case the authors emphazise
`combination system RecE of E; coll (48).
`that this repetitive sequence may provide the animal with genomic plasticity
`
`via mechanisms of DNA exchange.
`
`In fact,
`
`the kangeroo rat adapt rapidly to low
`
`levels of selection (48). This is what is observed also with the world-wide
`
`(high copy number of ClaI repeats) when compared
`distributed Eh; Eh; thummi
`with its endemic living relative §h;_th;_piger (low copy number). By the same
`argument, apparent alterations in the copy number of Clal repeats in thummi
`can account for a first step in species separation.
`
`ACKNOWLEDGEMENTS
`
`thank Prof. Dr. H.-G. Keyl for critical reading of the manuscript and
`I
`Dr. E. R. Schmidt who provided me with the cloned rDNA of Ch.
`th.
`thummi. The
`help and advice of Dr. E. A. Godwin in preparing of the manuscript is greatly
`acknowledged. This research was supported by the Deutsche Forschungsgeein-
`schaft, grant Ke 41, 15.
`
`*Dedicated to Prof. Dr. B. E. Holf in honour of his 75th birthday on
`September
`27, 1983
`
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