J. Cell Sci. 39, 63-76 (1979) 63Printed in Great Britain © Company of Biologists Limited

ORIGIN AND SPATIAL DISTRIBUTION OF

MATERNAL MESSENGER RNA DURING

OOGENESIS OF AN INSECT,

ONCOPELTUS FASCIATUS

DAVID G. CAPCO AND WILLIAM R. JEFFERY*

Department of Zoology, University of Texas, Austin, Texas 78712 U.S.A.

SUMMARY

In order to investigate the origin and spatial distribution of maternal mRNA during oogenesis,in situ hybridization with ['H]-poly(U) was utilized for the detection of poly(A)-containing RNA[poly(A) + RN A] in histological sections of Oncopeltus fasciatus ovaries. In the germariumpoly(A) + RNA was found to accumulate in the trophocyte cytoplasm concomitant with thematuration of these cells. Poly(A) + RNA was also detected in the trophic cores and nutritivetubes suggesting that these channels participate in the transport of trophocyte-derived mRNAto the oocytes. Although large amounts of poly(A) + RNA were also detected in the cytoplasmof the follicle cells, particularly during late vitellogenesis when pseudopod-like processesprojected into the ooplasm, no evidence was obtained for the transport of poly(A) + RNA fromthese processes to the oocytes. The content of poly(A) + RNA in the oocyte cytoplasm continuallyincreased during oogenesis. In stage 2-4 oocytes poly(A) + RNA accumulation occurred in theapparent absence of transcriptional activity in the germinal vesicle nuclei suggesting that mostmaternal mRNA molecules synthesized during early oogenesis are of trophocyte origin.Poly(A) + RNA also continued to accumulate after chorion formation, when the nutritivetubes are no longer active in RNA transport. This implies that other sources of maternalmRNA may exist during late oogenesis. The distribution of poly(A) + RNA molecules in theoocyte cytoplasm appeared to be uniform throughout oogenesis with one exception. Duringlate vitellogenesis poly(A) + RNA activity was significantly enhanced in the anterior andposterior periplasmic cytoplasms relative to the lateral periplasm and the endoplasm. Afterchorion formation these variations disappeared. The results suggest that maternal mRNAmolecules arise from at least 2 sources during oogenesis. During late vitellogenesis thesemolecules appear to be subject to differential localization in the polar perimeters of the oocytecytoplasm.

INTRODUCTION

The developmental patterns typical of mosaic eggs may be caused by morpho-genetic substances localized in the egg cytoplasm (Wilson, 1925; Davidson, 1976).Although the biochemical nature of these substances is unknown, the most likelycandidates appear to be informational macromolecules such as maternal mRNA andproteins (Whittaker, 1973, 1977; Kandler-Singer & Kalthoff, 1976; Jeffery & Capco,1978; Rodgers & Gross, 1978). If maternal mRNA and proteins are involved in thelocalization phenomena a differential distribution of these molecules in the egg cyto-plasm might be anticipated. In order to investigate the spatial distribution of maternal

• Author to whom correspondence should be addressed.5-2

64 D. G. Capco and W. R. Jeffery

mRNA in eggs we have developed a method for the detection of poly(A)-containingRNA [poly(A) + RNA], a class of mRNA (Brawerman, 1974), in histological sectionsby in situ hybridization with [3H]-poly(U) (Capco & Jeffery, 1978). Applying thismethod to the eggs of the milkweed bug, Oncopeltus fasdatus, we observed a uniformdistribution of poly(A) + RNA molecules in the cytoplasm prior to syncytial blasto-derm formation. In the present study we have extended our original application of the[3H]-poly(U) in situ hybridization technique to Oncopeltus ovaries. Our objectivewas to investigate the origin and spatial distribution of maternal mRNA duringoogenesis.

The meroistic ovaries of insects offer unique material to study the origin anddepositional pattern of maternal mRNA. Two types of accessory cells, the trophocytesand the follicle cells, as well as the oocyte genome itself are potential sites of trans-cription. In this type of ovary direct cytoplasmic connexions, which have been shownto function in inter-cellular transport of RNA (King, i960; Mahowald, 1973), existbetween the developing oocytes and the trophocytes. In this report we present evidencewhich suggests that maternal mRNA molecules originate from at least 2 sourcesduring oogenesis and that these molecules are subject to quantitative localization inthe polar cytoplasms of late vitellogenic oocytes.

MATERIALS AND METHODS

Cultures of Oncopeltus fasdatus were raised in plastic containers on a 14 h: 10 h, light:darkcycle as described by Beck, Edwards & Medler (1958). Ovaries were dissected from animalsin peak oviposition stage, about 2 weeks after their final moult. The ovarioles were separatedand fixed in absolute ethanol: acetic acid (3:1) for 15 min. The fixed ovarioles were dehydratedthrough a graded ethanol series, embedded in paraplast, and sectioned at 10 fim. In situhybridization with [7H]-poly(U) was carried out according to the procedure of Capco &Jeffery (1978) except that the concentration of [*H]-poly(U) applied to the sections wasi-8/tCi/ml (4-65 mCi/M; New England Nuclear Corp., Boston, Ma.). Briefly, slides werepretreated with 100 /Jg/ml DNase dissolved in 100 mM Tris-HCl (pH 7-6)~3 mM MgCL for1 h at 37 CC, annealing was performed in 10 mM Tris-HCl (pH 7-6)—200 mM NaCl-5 mMMgClj for 3 h at 50 °C, and after annealing the slides were successively rinsed with the hybridi-zation buffer and 50 mM Tris-HCl (pH 76)—100 mM KCl-i mM MgCla (TKM). UncomplexedPHJ-polyCU) was hydrolysed by treatment of the slides with 50 /tg/ml pancreatic ribonuclease(RNase) A dissolved in TKM for 1 h at 37 °C and removed by extraction with cold 5 %trichloroacetic acid for 10 min. Extracted slides were rinsed in distilled water, air-dried, andautoradiographed using Kodak NTB-2 liquid emulsion (Eastman-Kodak Special ProductsDivision, Rochester, N.Y.). Autoradiographs were exposed for 14 days, developed, and stainedthrough the emulsion with Harris haematoxylin-eosin.

RESULTS

Fig. 1 shows a longitudinal section through 2 Oncopeltus ovarioles subjected toin situ hybridization with pH]-poly(U). The dark areas on this low-magnificationmicrograph represent regions of intense labelling. The trophocytes at the anteriortip of the germarium and the follicle cells surrounding the oocytes are heavily labelledwhile the oocytes themselves exhibit less-intense labelling. The pH]-poly(U) bindingsites present in these ovariole sections, like those of Oncopeltus eggs and embryos

Origin and distribution of maternal mRNA *5

IFig. i. Poly(A) + RNA distribution in ovarioles of Oncopeltus fasciatus as determinedby in situ hybridization with [3H]-poly(U). Dark areas represent heavily labelled cellsof the germarium (#) and follicle cells (Jc) while the oocytes themselves are morelightly labelled, x 85.

66 D. G. Capco and W. R. Jejfery

Origin and distribution of maternal mRNA 67

(Capco & Jeffery, 1978), are selectively removed by pretreatment with RNase Adissolved in low-ionic-strength buffers suggesting they represent poly(A) sequences(Beers, i960; Darnell, Wall & Tuchinski, 1971).

Poly(A) + RNA distribution in the germarium

The distribution of grains seen in the germarium following pH]-poly(U) in situhybridization is shown in Figs. 2-5 and quantified in Table 1. Nuclear labellingappeared to be constant in trophocytes of all stages. However, the cytoplasmic grainswere more concentrated in mature zone III trophocytes (see Bonhag, 1955, for

Table 1. Concentration of grains over various regions of the Oncopeltusgermarium. following in situ hybridization with \^H]-poly(U)

Region

Trophocyte zone ITrophocyte zone IITrophocyte zone IIITrophic coreNutritive cord.Epithelial cells

Grain concentration represents the mean

Grain concentration(per io-/ima sample area)

Nucleus

146 ± 20n-3± 1-9IO-I ± 10

——

—

Cytoplasm

24-0 ±4-262-4±8-i

110-5 ± u"23i-3 ±4-730-0 ±4-3

9 0 ±1-4

number of grains ± standard deviation counted overio-/ims sample areas from 8 different ovarioles.

trophocyte staging) than in zone II trophocytes and the latter, in turn, exhibitedmore intense cytoplasmic labelling than zone I cells (Figs. 2-4). The overall increasein cytoplasmic labelling observed in zone III relative to zone I trophocytes wasabout 4-fold. These results suggest that poly(A) + RNA accumulates in the cytoplasmduring trophocyte maturation.

The trophic cores and nutritive tubes were also labelled following in situ hybridiza-tion, but their grain concentration was less than 30% of that found in the cytoplasmof the zone III trophocytes (Fig. 5; Table 1). Since nutritive tubes function in thetransport of trophocyte-derived products to the growing oocytes (Bonhag, 1955;MacGregor & Stebbings, 1970; Zinsmeister & Davenport, 1971; Davenport, 1976)the presence of poly(U)-binding sites in these structures implies that poly(A)+ RNA

Figs. 2-5. Poly(A) + RNA distribution in a germarium region of the ovary ofOncopeltus fasciattu as determined by in situ hybridization with [3H]-poly(U).

Fig. 2. Section through the anterior portion of a germarium showing labelledtrophocyte zones I, II, and III and part of the trophic core (tc). x 250.

Fig. 3. Section through the germarial region between zone I and II trophocytes.x 500.

Fig. 4. Section through the germarial region between zone II and III trophocytes.X700.

Fig. 5. Section through part of a nutritive tube (nt) and young oocytes (o). x 300.

68 D. G. Capco and W. R. Jeffery

may be among the transported substances. The low level of poly(U)-binding activityin the trophic cores and nutritive tubes relative to the zone III trophocyte cytoplasmcould mean that poly(A) + RNA transport through these structures is very rapid orthat the entire population of trophocyte poly(A) + RNA is not donated to the oocyte.The limiting factor in poly(A) + RNA transport may be related to the density ofcytoskeletal binding sites in the nutritive tubes.

Poly{A) + RNA distribution in the oocytes

As shown in Fig. 6, stage 2 oocytes (see Schreiner, 1977, for oocyte staging), theyoungest analysed, exhibited low levels of cytoplasmic labelling compared to thatfound in the trophocytes, trophic cores, or nutritive tubes. In more mature oocytesthe grain concentration per unit endoplasmic area gradually increased until a maximumwas reached at stage 5. Afterwards a steady decline in grain density began (Fig. 6).

50 -

- 20 „

3o

Figs. 6, 7. Poly(A) + RNA distribution in the endoplasm of Oncopeltus fasciatiisoocytes as determined by in situ hybridization with fH]-poly(U).

Fig. 6. Grain concentration per 100 fim1 endoplasmic counting area ± standarddeviation.

Fig. 7. Approximate endoplasmic grain number per total oocyte. The oocytestage terminology suggested by Schreiner (1977) is utilized with the exception ofstage 6" which cannot be distinguished from stage 6* by light microscopy.

Figs. 8—11. Poly(A) + RNA distribution in the cytoplasmic regions of Oncopeltusfasciatus oocytes as determined by in situ hybridization with [*H]-poly(U). Anterior,posterior, and lateral follicle cells are designated afc, pfc, and Ifc, respectively.

Fig. 8. Section through an endoplasmic region.Fig. 9. Section through the anterior polar periplasm.Fig. 10. Section through the posterior polar periplasm.Fig. 11. Section through a typical region of the lateral periplasm. All x 1500.

Origin and distribution of maternal mRNA

I fW***

70 D. G. Capco and W. R. Jeffery

The low grain concentrations seen in stage 7 oocytes approximated those previouslyobserved in freshly oviposited Oncopeltus eggs (Capco & Jeffery, 1978).

Since Oncopeltus oocytes increase markedly in volume during oogenesis due togrowth and yolk addition, the grain concentration per unit endoplasmic area is notrepresentative of the actual change in poly(U)-binding site number per oocyte. Whentotal oocyte labelling was approximated, by multiplying the endoplasmic grain numberof each total oocyte section by the estimated oocyte volume, it was found that the[3H]-poly(U)-binding site titre per oocyte continually increased during oogenesis,

e-jj

Fig. 12. The appearance of an Oncopeltus fasciatus stage 3 oocyte nucleus (arrow)following in situ hybridization with PHJ-polyCU). x 850.

with the largest increases seen between stages 6' and 7 (Fig. 7). The increase in[3H]-poly(U)-binding sites which occurs after the nutritive tubes have ceased transportof materials into the oocyte (Bonhag, 1955) suggests that post-vitellogenic oocytesaccumulate poly(A) + RNA molecules which originate from a source other than thetrophocytes.

In contrast to the oocyte cytoplasm, none of the nuclei examined during earlyoogenesis (stage 2-4 oocytes) showed significant labelling (Fig. 12). These resultssuggest that, at least in pre-vitellogenic and early vitellogenic oocytes, the oocytegenome may not be active in poly(A) + RNA formation.

The oocyte cytoplasmic regions were also scrutinized for differences in labellingfollowing [3H]-poly(U) in situ hybridization. In young (stage 2-5) oocytes grains wereuniformly distributed throughout the cytoplasm. However, when the endoplasmicgrain concentration began to be diluted by oocyte growth (Figs. 6-8), labelling in theanterior and posterior polar periplasms did not decrease and in some cases was

Origin and distribution of maternal mRNA 71

Table 2. Grain concentration in oocyte cytoplasmic regions of various Oncopeltusfasciatus ovarioles following in situ hybridization with [3H]-poly( U)

Oocytestage

Ovariole56'6 '7

OvarioleS6'6"7

Ovariole56'6 '7

Ovariole56'6 '7

Ovariole56'6*7

Ovariole56'6*7

Ovariole56'6*7

Ovariole56'6 '7

Anteriorperiplasm

no. 134'3 ± 4 °35'0±3743-2 ±3-8

9-i ± i ' 5no. 2

235 ±2-93i-2±5'827-5 ±2-3

2-5 ±i -9no. 3

3 2 - o ± 3 3

3 S - 2 ± 3 ' i357 ±3-°1 6 7 ± 2 0

no. 429-3 ±2-133-3 ±3-4222 ± 4 4

2 7 ± i-o

no. 522O±3'625-0 ±3-022-5 ±2'3

4-0 ± 1-4no. 6

38-0 ±5-23 6 7 ±2"O

53'5±4710-2 ± 6 0

no. 73 I O ± 2 ' I

30-5 ±4-237'5 ±5-°

5 ' 2 ± 1-2

no. 852O±3-6467 ±3'363-3 ±3'8

6-2± 12

Cytoplasmic grain

Posteriorperiplasm

51-216-4

Si-3 ± 3 34 6 0 ± 2 6

6 3 ± 1 2

33-8 ±2-540-0 ± 10525'O±2-5

3-012-3

527 ±4-°35'2±3'929 7 ±57I I - 8 ± I - 3

347 ±2-83S-2±3'8193 ±2-4o-8±o-8

45-0 ±4-03 i - 2 ± i - 5

23-3 ±3'43-o±i-S

6i-2±3'445'2 ± 1 7462 ± 7 07'S ±17

492 ±4943-8±5'i68-8 ±6-i3'8±o-8

60-814-9687 ±6-249'8 ±3'46-o±2-i

concentration

Lateralperiplasm

36-3 ±2733-0 ±373S-S±3'345 ± 1 °

300 ± 2 618-3 ±4-42O-O± 1-8

i -o ± 1 1

32-8 ± 2 3

2 9 - 5 ± 2 1

27-5 ±5-077±2-6

22-0 ± 7'O24-0 ±S'2I9-O ±3-53-2117

33 7±6-i10-7 ± 12I O - 8 ± I - 2

1-8 ± 1-2

35'2 ±3-2267 ±6-2263 ± 3 65'2±2-3

33-212-932-o±s-iI 6 - 2 ± 3 - I

4-2 ± i - S

53-0 ±4-522-2 ±2-O3 0 5 ± 3 14'S ±°-8

Endoplasm

5°-5 ±3941-813-4i8-2± i-83 7 ± 2 7

328 ±331 4 4 ± i ' ii 2 - 5 ± 3 - i

0-5 ±o-8

35'5±3-42 5 - 2 ± 3 0

2 i ' 3 ± 3 79-8 ± 2 3

2 1 7 ± 2 9

9'5 ±4'36-2 ± 1 2I -2±0 '8

32-7 ± 3 0

10-7 ±2-75-3 + 1-92 3 ± 1-9

32-8 ±2-426-7 ±2-5l 6 - 2 ± 2 47 '5±i-5

49-2±2-922-7 ±2'42 o o ± 1-54-8 ±1-7

767 ±4-8260 ±2-825-5 ±47

3'5 ±i '5

Grain concentrations are expressed as the mean grain numbers in 6 different ioo-/tm*areas ± standard deviation.

The periplasm is defined as the cytoplasmic region extending 40 fim inward from the edgeof the sectioned oocyte, while the endoplasm is defined as the cytoplasmic region internal tothe periplasm.

D. G. Capco and W. R. Jeffery

IT

4

Origin and distribution of maternal mRNA 73

intensified (Figs. 9-10; Table 2). The lateral periplasmic regions, unlike those at theoocyte poles, did not usually exhibit grain concentrations significantly different fromthe endoplasm (Fig. n ; Table 2). Although considerable variation in labellingoccurred between cytoplasmic regions of oocytes from different ovarioles, each stage6" oocyte examined showed about a 2-fold and sometimes a much greater enhancementof labelling in the polar periplasms relative to the endoplasm (Table 2). The variabilityof labelling seen in the polar periplasms of different ovarioles may be due to asyn-chronous growth of oocytes classified as the same developmental stage.

These results are consistent with the possibility that poly(A)+RNA molecules areenriched in the polar regions of the oocyte during late vitellogenesis. The polaraccumulations could not be detected in stage 7 oocytes.

Poly(A) + RNA distribution in the follicle cells

The follicle cell cytoplasm was also intensely labelled following in situ hybridizationwith pH]-poly(U) (Figs. 13-18). Labelling was particularly pronounced in the cyto-plasmic region adjacent to the oocyte surface at stages 6' and 6" (Figs. 13, 15). At thistime pseudopod-like projections, which have been previously described in thetelotrophic meroistic ovaries of Gerris (Eschenberg & Dunlap, 1966) and Rhodnius(Huebner & Anderson, 1972), were observed (Fig. 16). These projections sometimesappear as heavily labelled periplasmic spheres (Fig. 17), presumably due to sectioningthrough their knob-like termini. After chorion formation the projections disappearedbut the follicle cell cytoplasm remained labelled (Fig. 18).

DISCUSSION

In the meroistic ovaries of insects transcription of maternal mRNA, which iseventually deposited in the oocyte, could occur in the trophocytes, the follicle cells,or the oocyte nucleus itself (Mahowald, 1973). The present results, which have beenobtained by the use of in situ hybridization with pHJ-poh^U) for the detection ofpoly(A) + RNA (Capco & Jeffery, 1978; Jeffery & Capco, 1978), suggest that at least2 of these potential transcript sources donate maternal poly( A) + RNA to the Oncopeltusoocyte. Furthermore, a localized accumulation of maternal poly(A) + RNA has beendetected in the polar periplasmic regions of late vitellogenic oocytes.

During early oogenesis the trophocytes are probably the major source of oocytepoly(A)+RNA. Since the in situ hybridization procedure we have employed cannot

Figs. 13-18. Poly(A) + RNA distribution in the follicular epithelium of Oncopeltusfasciatus ovarioles as determined by in situ hybridization with PH^polyOJ).

Fig. 13. Follicle cells of stage 6' oocyte.Fig. 14. Interfollicular plug region (arrow) between stage 6" and stage 7 oocytes.Fig. 15. Follicle cells of stage 6* oocyte.Fig. 16. Follicle cell projections into the ooplasm of a stage 6* oocyte (arrows). This

section was not subjected to in situ hybridization with PHj-polyCU).Fig. 17. Heavily labelled follicle cell projections (arrows) into a stage 6" oocyte.Fig. 18. Follicle cells of stage 7 oocyte. All photomicrographs are x 750.

74 D. G. Capco and W. R. Jeffery

provide direct information on the transport of poly(A) + RNA, evidence for thetrophocyte origin of these molecules is based on the presence of poly(U)-binding sitesin the nutritive tubes, microtubule-containing corridors known to participate in RNAtransfer (Bier, 1963; Vanderberg, 1963; MacGregor & Stebbings, 1970; Zinsmeister &Davenport, 1971; Davenport, 1974, 1976). However, the possible contribution ofpoly(A) + RNA by follicle cells which border these passages cannot be excluded bythese data. Additional evidence for an extra-oocyte origin of early oogenetic maternalpoly(A) + RNA is provided by the lack of [3H]-poly(U)-binding sites in the nuclei ofstage 2-4 oocytes. The postulated trophocyte contribution of oocyte poly(A) + RNAis also consistent with our results showing that poly(A) + RNA accumulates in thecytoplasm of maturing trophocytes and the conclusions of radio-nuclide incorporationstudies recently carried out with silk moth ovaries (Paglia, Berry & Kastern,1976).

Following ligature of the nutritive tubes of Oncopeltus ovaries, Davenport (1976)detected very low levels of pHJuridine incorporation into high-molecular-weight,heterodisperse oocyte RNA and concluded that the oocyte nucleus may participatein maternal mRNA transcription during early oogenesis. Our results are not entirelyincompatible with the previous work since Davenport did not establish that RNAsynthesis actually occurred in the oocyte nucleus. Thus, the transcriptional activityobserved by Davenport (1976) could possibly be of mitochondrial origin. The ligationitself could also induce nuclear transcription in the oocyte. Alternatively, the absenceof appreciable [3H]-poly(U)-binding sites in the oocyte nucleus uncovered by ourstudies could also be explained by the masking of nuclear poly(A) sequences byproteins (Kwan & Brawerman, 1972) or other polynucleotide tracts (Jeffery &Brawerman, 1975).

It is clear from the present study that at least one other source of maternal mRNAbesides the trophocytes must exist since [3H]-poly(U)-binding sites markedly increasein quantity after the nutritive tubes are severed by chorion formation (Bonhag, 1955).The transcription of poly(A) + RNA in post-vitellogenic oocytes has also beendescribed in Dysdercus (Winter, Wiemann-Weiss & Duspiva, 1977). The identity ofthe other site(s) of oocyte maternal mRNA transcription in post-vitellogenic insectoocytes is currently unresolved. However, it is possible that the oocyte nucleus maybecome transcriptionally active at this time. Unfortunately, we could not obtainfavourable sections of Oncopeltus stage 7 oocyte nuclei to test this hypothesis.

The high concentration of [3H]-poly(U)-binding sites observed in the follicle cellcytoplasm suggests that it is rich in poly(A) + RNA. However, it is questionablewhether these molecules are transported into the oocyte as originally proposed byBier (1963) and by Vanderberg (1963). Our current observation of intense [3H]-poly(U)-binding activity within the follicle cell projections into the stage 6" oocytecytoplasm is not sufficient to suggest follicle cell RNA transfer since abnormallyhigh concentrations of binding sites were not observed on the oocyte sides of thesestructures. It is more likely that the follicle cell poly(A) + RNA molecules whichaccumulate proximal to the surface of stage 6* oocytes are involved in the precocioussynthesis of chorion proteins. Thus, we must conclude, as did Telfer (1964) previously,

Origin and distribution of maternal mRNA 75

that no evidence currently exists for the derivation of oocyte maternal mRNA from thefollicle cells.

A major objective of the present investigation was to test for the existence ofmaternal mRNA localizations in Oncopeltus oocytes. Our previous study, which alsoemployed in situ hybridization with pH]-poly(U) for poly(A) + RNA detection,suggested that such localizations were not present in freshly oviposited eggs (Capco &Jeffery, 1978). Our current results also suggest that oocyte poly(A) + RNA moleculesare uniformly distributed throughout the cytoplasm during most of oogenesis.However, one notable exception to this conclusion is the situation discovered in latevitellogenic oocytes in which accumulations of [3H]-poly(U)-binding sites appeared inthe anterior and posterior polar periplasms. If the differential distribution of thesebinding sites actually reflects variations in the titre of poly(A) + RNA molecules,rather than changes in relative length or accessibility of the poly(A) sequence itself,these findings imply that the polar periplasmic regions of late vitellogenic Oncopeltusoocytes are preferentially enriched in maternal mRNA.

Since the polar localizations of poly(A)+ RNA present in vitellogenic oocytes couldnot be detected after chorion formation they may be transient phenomena. However,masking or preferential turnover of their poly(A) sequences could also explain theirabsence in stage 7 oocytes and freshly oviposited egg9. In any case the activity of thepolar mRNA molecules could be instrumental in the construction of regional cyto-plasmic potentials which are of consequence during early embryogenesis.

We wish to express our appreciation to Dr Hugh S. Forrest for provision of Oncopeltusfasciatus cultures. Support for this work has been furnished in part by grants to W.R.J. fromthe NSF (PCM-77-24767) and PHS (GM26119).

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{Received 15 March 1979)