Analysis of genes for 5S rRNA from the cricket. Acheta domesticus: two classes of repeating units

113

Gene, 36 (1985) 113-122 Elsevier GENE 1309

Analysis of genes for 5s rRNA from the cricket, A&eta dome&us:

two classes of repeating units

(Recombinant DNA; Charon genomic library; SS rRNA genes; restriction and heteroduplex modulation of gene activity; amplification; oocytes; plasmid and A phage vectors)

mapping;

Helen Beneg*, Jerry Ware and M. Donald Cave Departmentof Anatomy, Universityof Arkansasfor Medical Sciences, Little Rock, AR 72205 (U.S.A.) Tel. (501) 661-5165 (Received October lOth, 1984) (Revision received February 4th, 1985) (Accepted February 1lth, 1985)

SUMMARY

To examine the modulation of 5s rRNA gene activity during development in the cricket, A&era domesticus, 5s +DNA was isolated from a IzCharon 4 genomic library and characterized. Southern blot analysis of cloned A. domesticus genomic DNA revealed that restriction fragments of 3.0 and 2.1 kb represent two size classes of 5s - DNA repeating units; over 90% of the repeats measure 3.0 kb. Restriction analysis of two 5s * DNA clones suggests that the 2.1-kb repeats are not randomly interspersed within clusters of the larger 3.0-kb repeating units. Heteroduplex and restriction mapping of several clones indicate that the spacers of both repeating units account for their unusual length. The major difference between the two classes of repeats may lie in 0.9-kb spacer sequences to the 3.0-kb repeats.

INTRODUCTION

In most animals, maturation of the oocyte involves the accumulation and storage of macromolecules and organelles which are used in the early stages of embryogenesis. There is, for example, in Xenopus

* Present address: Division of Gerontology Research, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (U.S.A.) Tel. (501) 661-5538. Abbreviations: AchSS, ICharon4AchSS clones; bp, base pairs; dNTP, deoxynucleotide triphosphate; Ch, Charon; DTT, dithiothreitol; EtBr, ethidium bromide; kb, kilobase pairs; p, plasmid; rDNA, DNA coding for 18S, 28s and 5.8s rRNA; 5s. DNA, DNA coding for 5s rRNA; rRNA, ribosomal RNA; SDS, sodium dodecyl sulfate; ss, single-stranded; SSC, 150 mM NaCl and 15 mM Na, . citrate, pH 7-8. 0378-I 119/85/%03.30 0 1985 Elsevier Science Publishers

Iaevis a loo-fold increase in the ribosome content

during the vitellogenic stage of oogenesis (Ford, 1971). The high level of rRNA gene activity, necessary for rapid ribosome assembly in the Xenopus oocyte, is ensured in part by amplification of oocyte rDNA sequences (encoding the 5.8S, 18s and 28s rRNAs) (Brown and Dawid, 1968; Gall, 1968; Brown and Blackler, 1972). The 5s rRNA genes, as well as the remainder of the Xenopus laevis oocyte genome, are not amplified (Brown and Dawid, 1968 ; Wegnez and Denis, 1972). Although the 5s rRNA genes greatly outnumber the rDNA repeating units in Xenopus somatic cells (respectively 22000 and 450 copies per haploid genome), in the mature oocyte the large rRNA genes are in 20-fold excess following rDNA amplification (Brown and Weber, 1968). Moreover, in Xenopus laevis about 90 % of the 22 000

114

5s rRNA genes code for an oocyte-specific 5s rRNA (Brown and Sugimoto, 1973; Brownlee et al., 1974; Peterson et al., 1980). Such tissue-specific heterogeneity of 5s rRNA resulting from differential gene expression has been demonstrated primarily in amphibians and fish. Another toad, Xenopus borealis, also exhibits rDNA amplification and expression of a specific 5s rRNA gene in the oocyte (Ford and Brown, 1976). In the teleost fish, Tinca tinca, there are different oocyte and somatic 5s rRNAs as well as rDNA amplification in the ovary (Denis and Wegnez, 1977). On the other hand, in a cartilaginous fish, rDNA amplification is absent, and 5s rRNAs from ovary and liver are identical (Wegnez et al., 1978). Recently, two 5s rRNAs have been described in somatic tissues of the chicken (Lazar et al., 1983). However, to date, tissue-specific expression of 5s * DNA has not been observed in insects (Benhamou et al., 1977). The structure and expression of rDNA in the house cricket, Acheta domesticus, have been the focus of a number of studies (Trendelenburg et al., 1973; 1976; Sharp, 1981). In particular, amplification of cricket rDNA sequences has been shown to proceed according to the amphibian model (Cave, 1973; 1979). We have undertaken a study of cricket 5s * DNA to determine what mechanisms might be involved in the control of Acheta 5s rRNA gene expression during development. In this report we present evidence for two sizes of cricket 5s * DNA repeating units which appear to differ primarily in their spacer sequences.

(b) Cloning of cricket 5s * DNA

A library of the A. domesticus genome was constructed according to Maniatis et al. (1978) by ligation of cricket DNA to the EcoRI sites of the arms of the phage, I Charon 4. For cloning, genomic DNA was extracted from cricket testes as previously described (Cave, 1979). All conditions for ligation, packaging, infection and plating were as described (Maniatis et al., 1978). The library consisted of 4.5 x lo5 phage/ml with an in vitro packaging efliciency of 7.5 x 105 phage/pg of target cricket DNA. Prior to amplification, the library was screened for the 5s rRNA genes, using the in situ plaque hybridization technique of Benton and Davies (1977) and a D. melanogaster 5s * DNA probe (plasmid 12D1, 32P-labeled by nick-translation). DNA from six putative 5s * DNA clones was prepared according to a modification of the method of Blattner et al. (1977). _&oRI fragments from the recombinant phage 1 Ch4-Ach5S4 (Ach5S4) were subcloned into pBR322 following the procedure of Mandel and Higa (1970) and Wensink et al. (1974). Bacterial colonies were screened according to a modification of the method of Grunstein and Hogness (1975). Hybridization conditions were as described below for restriction mapping with a 5s rRNA probe. Plasmid DNA was prepared either by the cleared lysate method (Clewell and Helinski, 1970) or a modification of the method of Bimboim and Doly (1979), followed by CsCl-EtBr-gradient purification. (c) Purification and labeling of cricket 5s rRNA

MATERIALS

AND METHODS

For identification and subsequent purification of A. domesticus 5s rRNA, a total nucleic acid extract

(a) Crickets, bacterial hosts, and Drosophila probe

Pre-wing adult house crickets, Acheta domesticus, were purchased from Fluker’s Cricket Farm, Baton Rouge, LA. Escherichia coli strains CSH18 and HBlOl (kindly supplied by Dr. F.C. Kafatos) were used for transfection and transformation. TheDrosophila 5s . DNAclone 12D1, constructed by Artavanis-Tsakonas et al. (1977) was generously provided by Drs. I. Cartwright and S.C. Elgin (Washington University, St. Louis) and served as a 5s . DNA probe.

of cricket testes was prepared. Testes from 4-5week-old male crickets were dissected in D. melanogaster Ringer’s medium and homogenized in RNA extraction buffer (10 mM Tris . HCl, pH 8.0, 1 mM EDTA, 0.35 M NaCl, 7 M urea, 2% SDS). Following extraction with phenol and chloroform-isoamyl alcohol (23 : l), the nucleic acids were precipitated in ethanol. Cricket 5s rRNA was identified by the Northern blotting technique (Alwine et al., 1977) using 2.25% agarose gels according to Bailey and Davidson (1979) and a 32P-labeled Drosophila 5s ’ DNA probe. Purification of cricket 5s rRNA

115

involved electrophoresis through preparative lowtemperature melting agarose gels under the same conditions. Methyhnercuric hydroxide could be omitted from preparative gels without any effect on the banding pattern. The band containing SS rRNA was excised from the gel and melted at 65 ‘C in an equal volume of extraction buffer (0.5 M NH, * acetate, 0.1% SDS, 0.1 M DTT). 5s rRNA was extracted twice with phenol and precipitated in ethanol. For use as a hybridization probe, A. domesticus 5S rRNA was 3’-labeled with 5’-[32P]cytidine 3’,5’bisphosphate (2000-4000 Ci/mmol, New England Nuclear) according to Peattie (1979). (d) Restriction analysis, DNA transfers, and labeling of DNA probes Following digestion with restriction endonucleases under conditions recommended by the supplier, fragments of genomic, plasmid or recombinant phage DNA were electrophoresed through an agarose gel in 40 mM Tris base, 5 mM Na * acetate, 1 mM EDTA (pH 7.9) and 1 pg EtBr/ml. The relative UV fluorescence intensities of EtBr-stained DNA bands were determined by scanning negatives at a wavelength of 540 nm. Transfer of DNA fragments from gels to nitrocellulose filters (Schleicher and Schuell) was performed according to Southern (1975). Either entire plasmids or isolated restriction fragments were labeled by nick-translation (Rigby et al., 1977) with one or more [ a-32P]dNTP (New England Nuclear). For analysis with a DNA or RNA probe, Southern blots were prehybridized in 4 x SSC, 4 x Denhardt’s solution (Denhardt, 1966), 250 pg/ml sheared calf thymus DNA, 0.1% SDS, 50% formamide at 42°C for 3 h. Hybridization was carried out overnight at the same temperature in 1 x Denhardt’s, formamide, 4 x ssc, 50% 100 pg/ml sheared calf thymus DNA, 0.1% SDS. When using DNA probes, incubations were also done at 65°C in the absence of formamide. Filters were extensively washed at 65 ‘C in a series of SSC solutions (4 x ,3 x ,2 x , 1 x SSC) with 0.1% SDS. Autoradiography was performed with Kodak XR-5 film and Cronex image intensifying screens at -70’ C. The relative intensities of autoradiographic signals were assessed from densitometric scans at 540 nm.

Restriction sites for Hi&I, AluI, HaeIII and Sau3A were mapped on isolated inserts of the plasmids pAch5SC118 and pAch5SC119 by the partial digestion technique (Smith and Bimstiel, 1976).

RESULTS

(a) Cloning of A. domesticus 5s +DNA To isolate 5S * DNA sequences, a genomic library of A. domesticus testis DNA was constructed in ACh4, and screened with the radiolabeled plasmid, 12D1, which contains 23 repeating units of D. melanogasfer 5S - DNA. Since the coding region represents over 30% of each D. melanogaster 5S - DNA repeat and 5S rRNA sequences are highly conserved among eukaryotes, strong homology was expected between A. domesticus and D. melanogaster 5S rRNA genes. Among the numerous 5S * DNA clones identified in screening the library, six were named according to Blattner et al. (1977): JCh4AchSS1,2, 3 . . . 6 and are referred to as AchSSl, 2, etc. DNA was prepared from each clone, digested with EcoRI, and fractionated by gel electrophoresis as shown in Fig. 1. Each 5S * DNA clone contains the expected Ch4 DNA fragments (the 20- and 1 1-kb arms) plus several EcoRI fragments of the cricket DNA. All six clones include a 3.0-kb EcoRI fragment, while AchSSl and Ach5S4 have a 2.1-kb fragment in common. To ascertain the location of 5S rRNA coding sequences among the A. domesticus EcoRI fragments of each clone, a Southern blot of this gel was probed with cricket 32P-labeled 5S rRNA. The autoradiogram (Fig. 1) indicates that only the 3.0and 2.1-kb fragments contain the 5S rRNA gene. According to the relative fluorescence of bands stained with EtBr (Fig. l), both the 3.0-kb and 2.1-kb EcoRI fragments occur as multiples within the cricket DNA inserts of the six clones examined. The weaker staining bands in clones Ach5S2, Ach5S3 and Ach5S5 may be DNA sequences flanking different clusters of 5S - DNA repeats. In situ hybridization of 5S rRNA to A. domesticus chromosomes revealed multiple clusters of 5S * DNA repeats (H.B. and M.D.C., submitted). To determine the number of copies of 3.0-kb and/or 2.1-kb EcoRI fragments in each Ch4 clone, each lane of an EtBr-

116 123456

123456

20

11

*

.

3.0

a

2.1

Fig. 1. Southern from

blot analysis

six A. domesticus

indicated

as

l-6)

of cricket

genomic

which

hybridized

5s. DNA probe was digested through

a 0.7%

fragments

DNA clones. DNA

(Ach5Sl to

with EcoRI

to Ach5S6,

a D. melunogaster and electrophoresed

gel (left panel).

to nitrocellulose

5s rRNA

and

(autoradiogram

hybridized

to

were

32P-labeled

in right panel).

stained gel (as in Fig. 1) was scanned using a spectrodensitometer. The number of copies of each A. domesticus EcoRI fragment was calculated, assuming that the ll- and 20-kb Ch4 arms (or their sum, appearing as a 3 1-kb fragment in the gel) occur only once per recombinant molecule. As shown in Table I, each cricket 5s * DNA clone contains at least three copies of the 3.0-kb EcoRI fragment which hybridizes to 5s rRNA. An additional two or three copies TABLE I Copy number cricket

of 5s

genomic

. DNA fragments

within the inserts

of live

clones

5S.DNA

Clones”

fragment

size” (kb) 1

2

Number

3

5

6

of copies per insertb

3.0

4.0

3.2

3.5

4.0

5.1

2.1

2.9

-

-

-

-

a Clones AchSSl, digested agarose

with

2, 3, 5 and 6 (indicated

EcoRI

and

as 1,2, 3, 5, 6) were through

a 0.7%

gel.

b The relative intensities determined number

electrophoresed

of the different EcoRI fragments

from densitometric

of copies was calculated

and 20 kb, occurring

(b) Length and organization 5s. DNA repeating units

of

A. domesticus

Sizes of the major

are given on the left in kb. The DNA fragments

transferred cricket

agarose

5s

clones

of a 2. I-kb fragment which hybridizes with 5s rRNA are found in two clones. For further analysis, the 3.0- and 2.1-kb EcoRI fragments of the genomic clone Ach5S4 were subcloned in the EcoRI site of pBR322. 5s *DNA recombinants were identified by colony hybridization to cricket 32P-labeled 5s rRNA. Plasmids were purified from three 5 S - DNA subclones (named pAch5SC118, -118, and -119, and referred to as ~118, pII8 and pII9), digested with EcoRI, and examined by Southern blot analysis. ~118 and pII9 each contain a 3.0-kb insert, while pII8 carries a 2.1-kb fragment ; only the inserts hybridize to A. domesticus 5s rRNA.

were

traces of each lane ofthe gel. The using the 2Ch4 fragments

only once per clone) as internal

(11

standards.

To determine the size of the 5s - DNA repeating unit, genomic blots of cricket testis DNA restricted to completion with EcoRI were probed for either the 3.0-kb or 2.1-kb EcoRI fragment using the plasmids pII8 and pII9 (Fig. 2). Both probes hybridized independently to 2.1-kb and 3.0-kb EcoRI fragments of A. domesticus genomic DNA. Furthermore, since in both cases the signal for the 3.0-kb fragment was much stronger than for the 2.1-kb fragment, these results can only indicate the existence of two different sizes of cricket 5s . DNA repeating units. The relative amounts of 3.0- and 2.1-kb repeating units per given amount of genomic DNA were assessed from densitometric scans of the autoradiograms (Fig. 2; H.B. and M.D.C., submitted). With the pII8 probe, the 2.1-kb repeats represent only 8.0% of the total 5s * DNA autoradiographic signal, whereas the pII9 signals indicate that the smaller repeats are contained within only 5.5 % of the 5s * DNA sequences. (This discrepancy can be explained by a lack of complete homology between the two repeats, as shown in the restriction and heteroduplex maps; see Figs. 5 and 6.) Thus, over 90% of A. domesticus 5s . DNA consists of 3.0-kb repeating units. Some of the possible arrangements of the two 5s - DNA repeats within the cricket genome were investigated using the Ch4 clones AchSSl and Ach5S6. Preliminary digestion experiments showed that the individual 5s . DNA repeats cloned within the plasmids pII8 and pII9 both contain only one BglII site, whereas a single Hue11 site is only found

117

1234

ized in tandem rather than in a head-to-head or tail-to-tail arrangement. Just how the two sizes of repeats might lie relative to one another within a cloned 5s * DNA cluster could be determined from Hue11 digestion of Ach5Sl DNA (Fig. 3). Restriction of Ach5S 1 DNA with Hue11 alone produced a 6.5-kb fragment which hybridized to the probe. This fragment must consist of two 2.1-kb repeats clustered at one end of the insert in addition to 2.2 kb from an adjacent 3.0-kb repeating unit. This result suggests that there are only two (not 2.9 or 3 as inferred from data in Table I) complete 2.1-kb repeats in the Ach5Sl clone. Maps of the 5s - DNA inserts of Ach5S 1 and Ach5S6 were constructed on the basis of the Hue11 and BgZII restriction experiments (Fig. 4).

567

4.3 3.0 2.1

(c) Restriction analysis of cloned 5s. DNA repeats

Fig. 2. Localization genomic

of 5s. DNA repeating

units in A. domesticus

DNA. 50 pg (lanes 3 and 5), 100 pg (lane 4) and 25 pg

(lane 7) of cricket

genomic

testis

DNA,

or the plasmids

(lanes 1 and 6) and pII9 (lane 2) were digested electrophoresed plasmid

through

DNA stained

and 6). The genomic hybridized

agarose

gel. The restricted

with EtBr served as markers DNA was blotted

to a 32P-labeled

pII9 (lane 7) probe.

a 0.7%

pII8

with EcoRI and (lanes

onto nitrocellulose

1, 2 and

pII8 (lanes 3,4 and 5) or 32P-labeled

Fragment

sizes are in kb.

in the 3.0-kb repeat (see Fig. 5). Therefore, DNA from the clones Ach5Sl and Ach5S6 was digested with BgZII or HueII, as well as with BglII + EcoRI or Hue11 + EcoRI. The resulting fragments were analyzed by Southern blot hybridization using the 32P-labeled 2.1-kb insert of the plasmid pII8 as a probe (Fig. 3). Given the sizes of the fragments which hybridize to the probe (in particular, the 2.4-, 1.5- and 0.6-kb fragments of the double BgZII or EcoRI digests of Ach5Sl), it is most probable that each 5s * DNA repeat within Ach5S6 and Ach5S 1 contains the unique BgZII site found in the cloned repeats of the plasmids 118 and 119 (Figs. 3 and 5). Furthermore, examination of the clone Ach5S6 suggests that each 3.0-kb repeat also contains a single Hue11 site. Both Hue11 and Bg111data indicate that A. domesticus 5s - DNA repeating units are organ-

To localize the 5s rRNA coding region within the 3.0-kb and 2.1-kb repeats ofA. domesticus 5s * DNA and also to compare these repeating units, they were mapped by restriction analysis. Of the 32 enzymes screened for their ability to cleave Ach5S4 or pII9 DNA, twelve (BumHI, BglI, HincII, HindIII, HpuI, KpnI, PvuII, SucI, SstI, SstII and XbaI) did not restrict cloned cricket 5s . DNA. Precise mapping of pII8 and pII9 was performed with the six enzymes shown in Fig. 5. At each step in the mapping strategy, the digestion products were examined by agarose gel electrophoresis and Southern blot analysis using a radiolabeled A. domesticus 5s rRNA probe. The numerous restriction fragments obtained with certain enzymes were placed using the partial digestion technique. Fig. 5 summarizes the restriction mapping of cloned examples of the two sizes of cricket 5s * DNA repeating units. The 5s * DNA inserts of the plasmids pII8 and pII9 lie in opposite orientations with respect to the Hind111 site of pBR322. The 5s rRNA coding region is localized to one end in both the 3.0- and 2.1-kb repeats, specifically to a region within 0.30 kb of the EcoRI site delimiting each repeat. Hybridization of restriction fragments to A. domesticus 5s rRNA consistently showed that the 0.52- (or 0.55-) kb HintI -EcoRI fragment of each repeat represents the only portion which contains any 5s rRNA coding sequences. Since two Suu3A fragments (0.20 and 0.37 kb) hybridize to 5s rRNA,

118

AB

CD

EF

GH

3.0 3.6 2.2

2.4 2x

0.6 o

0.95

.6 0.95

Fig. 3. Restriction analysis of two A. domestics 5s. DNA clones. DNA from clones Ach5S6 (A-D) and AchSSl (E-H) was restricted with BglII (A, E), BglII + EcoRI (B, F), Hue11 (D, H) or Hue11 + EcoRI (C, G) and electrophoresed through a 1.2% agarose gel. The DNA fragments were transferred to nitrocellulose and hybridized to the 32P-labeled Xl-kb insert of the plasmid pIIS. Autoradiograms lie to the right in each panei; fragment sizes are in kb.

P

P

TP

TP

TP

TP

TP

TP

TP

TP

TP

7

?

P

Fig. 4. Organization of cricket 55 . DNA repeats in genomic clones AchSSl (upper) and AchSS6 (lower). The A-J arm (left) and N-R arm (right) of ICh4 DNA sequences are represented by thick black lines. The thin lines represent cricket 5S - DNA. Restriction sites for EcoRI (arrows), Bg1II (open squares) and Hue11 (closed squares) are indicated. Figure is drawn to scale.

it is still possible that the 0.52-kb IfiHfI-EcoRI fragment harbors more than one coding region. However, Fig. 5 depicts the simpler explanation of Suu3A cleavage of a single 5s rRNA coding region. The presence of a single 120-bp coding region would imply that over 90% of both the 2.1- and 3.0-kb repeat units consists of spacer DNA. As shown by the dashed lines in Fig. 5, homologous positions for all the restriction sites determined for the 2.1-kb insert of pII8 can be found in the 3.0”kb insert of

~119. The major difference between these cloned A. do~e~~ieu~ DNA repeating units, one which can be detected by restriction mapping, lies in the central portion of the spacer of the 3.0-kb repeat. This region is indicated by a bracket in Fig. 5 and is crudely delimited by Sau3A sites. Not only does this DNA sequence correspond approximately to the difIerence in length between the 2.1- and 3.0-kb repeats, but it includes the Hue11 site absent in the 2.1-kb unit.

P

Hinf I -f . Hoe III -

I,

Bglll-0,

HoelI-!,

5s ’ DNA inserts of the recombinant plasmids Fig. 5. Restriction maps oftwo cloned cricket 5s 1DNA repeating units. TheA. dD~esti~ pi18 and pII9 were mapped using the indicated enzymes. The thick black lines and the thin lines represent pBR322 and cricket 5s *DNA sequences, respectively. The open box designates the putative position of 5S rRNA coding sequences. The dashed lines indicate possible homologous restriction sites whereas the horizontal bracket points to 5s . DNA spacer in pII9 not observed in ~118.

(d) Heterodupiex 55 - DNA repeats

analysis

of

cloned

Acktitu

Given the unexpected similarities found in the restriction maps of the 5S * DNA plasmids pII8 and pII9, the extent of homology shared by the two cloned repeating units was examined by electron microscopy of spread DNA. The 3.0- and 2.1-kb 5S - DNA fragments were isolated from their respective plasmids, denatured and allowed to reanneal under conditions favoring heteroduplex formation (Fig. 6). Heteroduplexed molecules exhibited a daublestranded Iength of 1.94 + 0.08 kb (n = 36). An ss loop of 0.95 kb +, 0.12 (n = 36) corresponding to spacer DNA sequences of the pII insert was localized 0.61 2 0.08 kb (a = 36) from one end of the heteroduplex. The placement of the ss loop, at 0.61 kb from one end of the pII8 insert, confirms the designation by restriction mapping of this nonhomologous region (see bracket in Fig. 5). Indeed, given the discrepancy in measurements by two different t~h~ques (gel e~~~ophoresis and eiectron microscopy) the 0.95kb loop also corresponds to

the difference in length between the two repeating units.

Fig. 6. Three examples of heterodupiexes between the 5s . DNA inserts of the plasmids pII8 and ~119. Heteroduplex molecules were formed (Westmorel~d et al., 1969; Ferguson and Davis, 1978) by reannealing 2.5 &ml of the pII insert with the same concentration of the pII9 insert, Circular pBR322 was added as a length marker (example in lower part of left panel), and a 1:20 dilution in 70% Formamide was spread by the procedure of Rleinschmidt and Zahn (1959). DNA molecules were photographed through a Siemens 1-A electron microscope at an initial magnification of 24000 x . A noncomplementa~ region, represented by a ss loop rne~u~ng 0.95 kb (X,) was localized 0.61 kb (X,) from one end of the 5s. DNA insert of ~119.

120 DISCUSSION

DNA content (2 pg or 1.93 x lo9 bp) for A. domesticus (Lima de Faria et al., 1973).

Isolation of A. dome&us 5s * DNA by molecular cloning produced two EcoRI fragments, measuring 3.0 and 2.1 kb, which were subsequently shown to be representatives of two classes of 5s * DNA repeats. These repeating units are the largest reported for any eukaryote whose rDNA and 5s - DNA sequences are organized in separate clusters of repeats. Large 5s * DNA repeating units, measuring 1.2 and 1.3 kb in length, have been described for the sea urchin, Lytechinus vurkgatzu (Lu et al., 1981). Unusually long repeating units may be a particular characteristic of the organization of all four cricket rRNA genes. According to restriction analysis and electron microscopy, the A. domesticus rDNA repeating unit spans over 54 kb (Trendelenburg et al., 1976; Cave, 1979; Z.D. Sharp, J. Ware and M.D. Cave, submitted) while, in contrast, the 18s and 28s rRNA genes of the sea urchin lie within 11-kb repeating units, an average size for most higher eukaryotes (Blin et al., 1979). Furthermore, the difference in length (about 0.9 kb) between the two cricket 5s * DNA repeats is most striking. In most organisms studied to date, the classes of 5s * DNA repeats commonly differ by 10 to 100 bp, while differences up to 600 bp characterize the oocyte-specific and somatic-specific repeats of X. luevti. Although pseudogenes adjacent to intact 5s rRNA genes have been described in amphibian 5s * DNA (Jacq et al., 1977; Kay and Gall, 1981), the presence of pseudogenes cannot explain the difference in length between the two classes of cricket 5s * DNA repeats. One repeating unit length tends to predominate in organisms exhibiting two size classes of 5s * DNA repeats. In X. laevis the oocyte-specific repeats contain over 90% of the 5s rRNA genes (Ford and Brown, 1976). The major repeating unit in the newt also accounts for over 90% of that amphibian’s 5s * DNA (Kay and Gall, 198 1). In wheat and flax, the smaller repeat constitutes about 80% of the total 5s * DNA, and the larger repeating unit may not even be transcribed in both of these plants (Gerlach and Dyer, 1980; Goldsbrough et al., 1981). Here we report that the 3.0-kb repeating units represent over 90% of A. domesticus 5s * DNA sequences. In fact, the difficulty in detecting the 2.1-kb repeats on genomic blots (up to 100 pg of genomic DNA probed in Fig. 2) may be due to a relatively high haploid

Examination of the genomic organization of cricket 5s * DNA in six clones suggests that there may be long stretches of cricket 5s . DNA harboring only tandemly repeated 3.0-kb units, while mapping of the insert of the AchSSl clone shows that the 2.1-kb repeats may be tandemly clustered within a stretch of the larger repeating units. Since chromosomal localization ofA. domesticus 5s * DNA sequences by in situ hybridization demonstrated several clusters of repeats (H.B. and M.D.C., submitted), the clustering of 2. I-kb repeats may be similar to the organization of somatic-specific repeating units in X. faevis. Harper et al. (1983) have recently shown that the X. laevis somatic-specific repeats occupy only one chromosomal locus within a cluster of oocytespecific repeats which are distributed on the telomeres of all the chromosomes. Sea urchin 5s * DNA is also arranged as two tandemly, noninterspersed families which may result in tissuespecific expression of 5s rRNA (Blin et al., 1979). In contrast, in the newt the two sizes of repeating units may not represent ditferent types of genes and are organized within stretches of homogeneous and interspersed repeats (Kay and Gall, 1981). Comparison of representatives of the two size classes ofA. domesticus 5s 1DNA repeats by restriction and heteroduplex analysis revealed that the spacers account for the major difference observed between these repeat units. Sequences within 1 kb of the EcoRI site and including one or more coding regions exhibit very strong homology, while a 0.9-kb region in the spacer of the larger repeat produces both sequence and length heterogeneity. With the recent attention given to mobile genetic elements in the control of eukaryotic gene expression, it will be valuable to examine the 0.9-kb region as a potential transposable element. By comparing the two 5s *DNA repeats of A. domesticus, one may also consider whether these repeating units might not be components of a dual 5s rRNA gene system, as may be characteristic of organisms whose rDNA is amplified during oogenesis. The analysis reported here suggests certain features for A. domesticus 5s - DNA which distinguishes it from the somatic oocyte and specific repeating units of X. luevis. Whereas virtually no complementarity can be demonstrated between the

121

X. lueti somatic- and oocyte-specific spacers, spacers from both classes of A. domescicus repeats exhibit extensive homology. Yet the two sizes of cricket 5s * DNA repeats may not be a simple case of length heterogeneity in a single type of repeating unit. In both rDNA and 5s * DNA of several organisms, variable numbers of internal subrepeats within the spacer are a source of length heterogeneity (Long and Dawid, 1980; Tschudi and Pirrotta, 1980; Long et al., 198 1). Subrepeats remain to be demonstrated in the A. domesticus 5s - DNA repeats, as they are not evident from the restriction analysis. Indeed, the question of a dual 5s rRNA gene system in the cricket, as well as full characterization of differences between the two classes of repeats, will have to await DNA sequence analysis.

ACKNOWLEDGEMENTS

This paper is based on a dissertation submitted by H.B. in partial fulfiient of the requirements for the Ph.D. degree at the University of Arkansas for Medical Sciences. The work has been supported by a grant to H.B. from the Graduate School, University of Arkansas for Medical Sciences. We are very grateful to Fotis C. Kafatos and Jack Crawford for the use of their laboratory facilities during the cloning experiments. We especially thank Thomas H. Eickbush for participation in the construction and screening of the library and all subsequent advice. Jim Hardin and Bob S. Reis generously provided helpful discussions as well as gifts of enzymes and radiolabeled nucleotides. We also gratefully acknowledge Jean-Antoine Lepesant and Susan A. Gerbi for critical reading of the manuscript, and Yvonne Wingfield for expert typing.

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