juvenile hormone action to suppress gene transcription and influence message stability

DEVELOPMENTAL GENETICS 14323332 (1993)

Juvenile Hormone Action to Suppress Gene Transcription and Influence Message Stability GRACE JONES, VENKATESWAR VENKATARAMAN, MARIA MANCZAK, AND DOUGLAS SCHELLING School of Biological Sciences, University of Kentucky, k i n g t o n

ABSTRACT Proteins normally expressed in high abundance only at lawal-pupal metamorphosis in Trichoplusia ni were examined in a compar- ative analysis of the role and level of hormonal control of their expression. Some related proteins in the hemocyanin-superfamily (i.e., an acidic protein [AJHSPl] and two basic proteins [BJHSPl, BJHSP21) were shown by nuclear run-on analysis to be specifically transcriptionally suppressed by juvenile hormone (JH), while transcription of another member of that family which is also metamorphosis-associated (arylphorin) was not specifically sensitive to JH. The stability of the mRNA for those members transcriptionally down-regulated by JH appeared to decrease under high JH conditions. While each protein was resorbed to some extent by the prepupal fat body, only the two basic proteins were quantitatively cleared from prepupal hemolymph. The JH-sensitive proteins studied appear to be encoded in single copy genes not immediately juxtaposed in the genome. These and previous studies now permit a more comprehensive understanding of the different combinations of mechanisms involving transcription, mRNA stability, translation, and protein clearance that operate to regulate these metamorphosis-associated proteins. 0 1993 Wiley-Liss, Inc.

Key words: mRNA stability, mRNA translatability, metamorphosis, gene regulation, hexamerins, storage proteins

INTRODUCTION The mechanisms by which gene expression are reg-

ulated so as to achieve precise temporal specificity is a fundamental question in developmental biology. Dur- ing insect development through the egg, larval, pupal, and adult forms the organism goes through several dis- crete stages with radically different body form. This feat requires precise regulation of stage-specific patterns of gene expression.

Proper expression of at least part of the gene pattern associated with insect metamorphosis requires a decline in juvenile hormone IRiddiford, 19801. Conse- quent to this decline, a number of genes increase dra- matically in expression or become detectably expressed

for the first time. Examples of JH-sensitive genes whose expression is associated with metamorphosis are certain cuticle genes [Hiruma et al., 1991; Roberts and Willis, 19801 and certain fat body-produced hemolymph proteins such as vitellogenin and hexamerins [Wyatt, 1991; Kanost et al., 1990; Telfer and Kunkel, 19911. In these examples, the abundance of mRNA for the protein is regulated by JH.

“he molecular mechanisms by which J H regulates the metamorphic specificity of the pattern of expression of these proteins are not well understood [Laufer and Borst, 1983; Riddiford, 1985; Willis, 19741. Some nuclear proteins which bind to JH-photoaffinity labels have been identified [Palli et al., 1991; Prestwich, 1991; Shemshedini et al., 19901. However, identification of an insect J H receptor as a transcriptional regulator which acts in a manner similar to steroid or retinoic acid re- ceptors has remained elusive. Although some JH-response elements have been proposed for adult or- thopteran protein genes, on the basis of comparison of 5’ flanking sequences [Wyatt, 19901, no JH response elements have been proposed for any other insect. Fur- ther, while mRNA abundance has been shown to re- spond to JH treatment, an influence of JH on actual transcription of these genes has only been indirectly inferred. Thus, although JH or JH-like molecules are considered to be major regulators of gene expression in invertebrates, there has never been a direct demonstration that JH actually affects gene transcription.

We have identified in a lepidopteran, Trichoplusia ni, several proteins whose dramatic increase in abundance in the hemolymph is strictly associated with ini- tiation of larval-pupal metamorphosis [Jones et al., 1987bl. This increase in abundance is also suppressible by JH [Jones et al., 1988, 1987aI. These proteins are members of the large group of hemocyanin-related proteins, in which various families of proteins exist [“hexamerins,” Telfer et al., 1983; Telfer and Kunkel, 19911. We have also identified proteins in the hemocyanin- group whose hemolymph abundance is not sensitive to

Received for publication January 14, 1993; accepted April 12, 1993.

Address reprint requests to Grace Jones, School of Biological Sciences, University of Kentucky, Lexington, KY 40506.

0 1993 WILEY-LISS, INC.

324 JONES ET AL.

juvenile hormone (e.g., arylphorin). These proteins and their cognate genes thus offer a rich source of material for identifying mechanisms and levels of regulation of genes associated with immature to adult differentiation in invertebrates. We report here nuclear run-on analysis of the marked temporal changes in rates of transcription of these genes during metamorphosis. We also present the first demonstration that suppressed transcription of these genes is the basis for the previously reported suppression of their transcript abundance. Finally, additional data reported here and elsewhere [Jones et al., 19931 together provide evidence for stability as a level of regulation of their transcript abundance.

MATERIALS AND METHODS Insects and Hormone Treatment

Larvae of T. nti were reared and developmentally staged during the final 4-day stadium as previously reported [Jones et aZ., 19811. Larvae were topically treated at the end of the penultimate stadium (48 hr prior to day 1 of the final stadium) with 10 nmol of fenoxycarb (Hoffman-LaRoche) in ethanol, a stable JH analog shown to cause specific JH-like effects in this insect [Jones, 19851. This analog shows a dose-depen- dent effect between 10 pmol and 10 nmol in suppress- ing the same metamorphosis-associated proteins (e.g., the 73-76 kDA proteins studied here) that are specifically suppressed by natural juvenile hormone [Jones et al., 19881. This analog also suppresses this group of 73-76 kDa proteins when applied directly to the fat body in vitro [Jones et al., 1987al. The dose of hormone analog used here sufficient to cause the treated larvae to undergo apolysis to a JH analog-induced extra larval molt 5 or more days after treatment [Jones, 19851. Nu- clei were extracted from ca. 30 larvae for each treatment and time point on days 1, 2, 3, or 4 post-treatment.

Nucleic Acid Extraction Poly(A) RNA was extracted from T. ni and purified

by oligo (dT) cellulose chromatography as described [LeMeur et aZ., 19811. Genomic DNA was prepared by methods described elsewhere [Strauss, 19911.

Nuclear Run-on Transcriptional Assay Nuclei were isolated from fat bodies of ca. 30 larvae

for each measurement by a procedure similar to that described by Abmayr [19911, through the step of isola- tion of nuclei and stored until use. The transcription reaction was carried out as per Guertin et al. [19831, except for a slightly lower Mg++ concentration in a total volume of 150 p1 containing 20 mM Hepes-NaOH (pH 7.5), 150 mM NaC1, 5 mM Mg acetate, 0.5 mM MnCl,, 2 mM DTT, 0.4 mm each of ATP, CTP, and GTP, 500 U/ml RNAsin, 1 mM creatine phosphate, 3 U/ml of creatine phosphokinase, 50 pCi of a l ~ h a - ~ ' P

UTP (600 Ci/mmol), 16% glycerol and an amount of nuclei corresponding to 200 pg of nuclear DNA. After a 10 min incubation at 26°C the labeled RNA was purified by hot-phenol extraction, made 0.2 M Na acetate (pH 4.41, 1% SDS, and an equal volume of phenol added. After incubation for 5 min at 55"C, it was chilled on ice for 5 rnin and centrifuged at 10,OOOg for 10 min at 4°C. After chloroform extraction and two ethanol precipitations the RNA pellet was dissolved in water, spotted to Whatman GF/C filters, and washed three times for 10 rnin in 5% TCA. The spots were scintillation counted to estimate total transcription (total incorporated radiolabel).

Transcription of specific RNAs was measured by hybrid selection of transcripts with 10 pg of cDNA for the transcript of interest. Briefly, cDNA encoding a gene of interest, and vector plasmid DNA as a control, were denatured with 0.4 M NaOH and then raised to 1 M Na acetate and spotted to nitrocellulose (prerinsed with 1 M NH, acetate). The filters were washed with 6 x SSC, UV crosslinked, and prehybridized for 12 hr a t 65°C (hybridization solution: 0.5 M Na phosphate pH 7.5,1% BSA, 1% SDS and 200 pg/ml yeast RNA). The filters were then placed in hybridization solution containing the labeled RNA and incubated for 72 hr at 65°C. After washing three times at 65°C with 40 mM NaPO, containing 1% SDS, the filters were treated with 10 Fgiml RNase A at room temperature for 20 min to remove all nonspecific hybridization. After three more washes in 40 mM NaPO,, the filters were scintillation counted.

Calculation of Rates of Transcription The rate of transcription was examined in several

ways so as to answer different questions on the nature of the temporal or hormonally induced changes in transcription. We designed the experiments to test whether juvenile hormone caused changes in transcription of particular genes by a general action on all genes or by a specific action on the gene of interest.

First, the question of changes in the rate of total genomic and of gene-specific transcription was addressed by measurement of the total 32P-UTP incorporated in the run-on reaction, and of the 32P-UTP incorporated into transcripts for each particular gene. These measurements enable determination of the total genomic transcriptional synthesis during the reaction from 200 pg of nuclear DNA, as well as the total transcriptional synthesis from particular genes.

The second question addressed was on the relative rate of transcription of particular genes as a proportion of total nuclear transcription. To obtain such measurements, an equal number of incorporated cpm (50,000) were taken from each transcription reaction for hybridization to each specific probe. This method eliminates the contribution to changes in transcription of specific genes that is due to general, non-specific changes across the genome as a whole. Thus, this measurement

JH ACTION TO SUPPRESS GENE TRANSCRIPTION 325

is assessing the relative transcription of a particular gene as a percentage of total genomic transcription.

Protein Preparation and Analysis Proteins were extracted from dissected fat body by

homogenization in 50 mM Na phosphate, pH 7.4, and protein concentration was determined with Bio-Rad re- agent (Bio-Rad). Hemolymph was taken from at least 3 larvae per time point and the protein concentration similarly determined. For each time point, equal amounts of protein for each time point for fat body protein (30 pg) or equal amounts of hemolymph (1 p1) were used. The proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis [Laemmli, 19701 and visualized by either silver staining [Blum et al., 19871 or immunoblotting [Burnette, 19811. Characterization of antisera used for the immunoblots is found in Jones et al. 119881 and Jones et al. 119931.

cDNA Clones The clones used as probes for measurement of mRNA

abundance, or for nuclear run-on analysis of transcription were as follows: a full-length cDNA for an acidic, JH-suppressible protein [AJHSPl, Jones et al., 19901; full-length cDNAs for two related basic, JH-suppressible proteins TBJHSP1, BJHSPB, Jones et al., 198733, 19931; and cDNA for arylphorin [Jones et al., 19931.

DNA Sequencing Cloned cDNA was sequenced by the method of

dideoxy chain terminators [Sanger et al., 19771.

RESULTS Developmental Expression of Total

Nuclear Transcription The rate of total nuclear transcriptional activity dur-

ing larval metamorphosis was measured to provide a baseline against which the transcription of specific genes could be compared. As shown in Figure la , this rate of total genomic transcriptional activity was essentially constant and high during the first 2 days (feeding stage) of the final stadium. Then, the rate took a precipitous drop on day 3 (nonfeeding, wandering stage), to a low level maintained through day 4 (nonfeeding, prepupal stage). Hormone-treated larvae also showed a sharp drop in the rate of total transcription on day 3, even though the hormonal treatment ex- tended the feeding stage through all 4 days.

Transcription of Specific Genes During Metamorphic Development

Rate of transcription. The transcriptional activity of specific genes was measured to determine whether their changes in transcription were similar and whether such changes generally paralleled changes in total nuclear transcription. The pattern of developmental change in the transcription rate occurring for each

of the four specific genes was similar during the final stadium, except for the arylphorin. Each gene showed a ca. %fold increase during the 2 day feeding stage, and then a decline to a lower, essentially constant level on days 3 and 4 (Fig. lc-e). In contrast to the other genes, arylphorin transcription occurred at a much higher and constant rate during the first two days of the final stadium, before a sharp decline on day 3 and a return to a high rate on day 4 (Fig. lb).

For AJHSP1, BJHSPl, and BJHSP2, the hormone treatment had a distinct suppressive effect on the rates of their transcription on day 2, but had little effect on days 3 and 4 larvae as compared to untreated larvae (Fig. lc-e). Hormone treatment caused an initially lower (day 1) but then higher (day 2) than normal rate of transcription of arylphorin (Fig. lb). Finally, the rate of arylphorin transcription in hormone-treated larvae was below normal by day 4.

Relative transcriptional activity. The effect of hormone treatment on the rate of transcription of particular genes relative to the total transcription of all active genes was measured on each day of the final stadium. Each specific gene normally showed an increase in its relative transcription rate through days 1-3, and then a decline on day 4, except for arylphorin (Fig. 2 M ) . Arylphorin showed a continued increase in relative transcription through days 1-4 (Fig. 2a). Hor- mone treatment caused a distinct suppression of relative transcription rates of all genes, except arylphorin, on days 2 and 3. Except for arylphorin, the hormonal effect on each specific gene by day 4 was a relative level of transcription higher than the maximum normal rate observed on any day.

Specificity of hormonal effect on transcription rate. Analysis was made of the specificity of the hormonal effect on a particular gene above and beyond the average hormonal effect across all active genes. This analysis was made by comparing the ratio of the absolute rate of transcription of a specific gene of interest in [hormone-treatedl/lcontrol larvae1 to the ratio calcu- lated for total nuclear transcription of all active genes in [hormone-treatedl/[control larvael. AJHSP1, BJHSP1, and BJHSP2 exhibited a clear specific suppression in transcription on day 2 due to hormone- treatment, while in contrast total nuclear transcription was increasing in these hormone-treated larvae. In contrast, the pattern of change in the ratio for arylphorin across all four days exactly followed the change in pattern for total nuclear transcription, that is, high JH was not specifically affecting arylphorin in a way different than the “average” effect across all active genes.

Developmental Pattern of Fat Body and Hemolymph Content of

Hemocyanin-related Proteins Since the transcription and biosynthesis of AJHSPl,

BJHSP1, BJHSP2 and arylphorin are known to occur

326 JONES ET AL.

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Fig. 1. a) Total nuclear transcriptional activity, and transcription of specific genes, as influenced by developmental stage and juvenile hormone. Total transcription (expressed as cpm) by nuclear DNA equivalents on each day of the normally 4-day long final stadium (solid line) and on days 1-4 of final instar larvae treated at the final larval molt with a J H analog (dotted line). b-e) Total transcription of specific genes in normal and JH-treated final instar larvae. The specific genes analyzed in each panel are indicated by the abbreviation or acronym shown. Measured cpm of J*P UTP incorporated has been normalized to equivalent amounts of nuclear DNA in each run-on assay for each day. The pattern of changes in incorporated cpm shown indicate changes in the absolute magnitude of transcription of the given gene. The two data points at each time are shown as squares, with their means shown as dots.


1 2 3 4


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Fig. 2. Transcription of specific genes, as a proportion of total genomic transcription by juvenile hormone. Transcription is shown for days 1-4 of the final stadium in normal (solid line) and JH analog- treated (dotted line) larvae. The specific gene analyzed is designated

(primarily) in the fat body followed by release into the hemolymph [Palli and Locke, 1987a,b; Jones et al., 19931, it was relevant to measure the developmental pattern of their abundance in that tissue, in comparison with their transcription. As shown in Figure 3, the proteins displayed different developmental patterns of abundance. The two basic proteins (BJHSP1, BJHSP2) were not detectable on day 1, were weakly detectable on days 2 and 3, but very highly abundant in the fat body on day 4. Concomitant with their marked day 4 increase in the fat body (and their sharply reduced transcription), the two basic proteins quantitatively disappeared from the hemolymph.

The profile of fat body content of AJHSPl was similar to that for the two basic proteins in that the day 1 abundance was very low, days 2 and 3 were higher, and day 4 the highest. However, the difference between the days 2 ,3 , and day 4 abundances was not as great as for the basic proteins. Markedly different from the basic proteins was that AJHSPl remained in high abundance in the hemolymph on day 4 relative to other

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uted by the specific gene (see Materials and Methods).

proteins (despite the decline in its relative rate of tnan- scription).

Finally, the pattern for arylphorin was different in that after the rise in its abundance in the fat body and hemolymph from day 1 to day 2, it showed a marked decrease in abundance in the fat body on day 3 (Fig. 3) in parallel with the disappearance on day 3 of its transcript [Jones et al., 19931 and of its biosynthesis (35S-met incorporation experiments not shown). Yet it remained at a very high concentration in the hemolymph, and continued to remain high there on day 4, while also its abundance in the fat body rose on that day.

Genomic copies of genes. Hybridization with genomic DNA was performed to assess the relationships among the hormonally sensitive proteins with respect to genomic organization. At the level of resolution used, the results shown in Figure 5 do not suggest that each gene is present in the genome as more than a single copy. In addition, the genomic restriction patterns associated with the genes did not suggest an im-

328 JONES ET AL.

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

B JHSP1 BJHSP2

Fig. 3. Developmental changes in abundance of specific proteins in the fat body (top) and hemolymph (bottom) on each day of the final larval stadium. Proteins were probed with antisera against the specific indicated protein. Hemolymph samples were taken at two times

BJHSP1 BJHSP2 AJHSP1 5 4- 3-

kb 2-

1-

Fig. 4. Genomic Southern blot analysis of genes for AJHSP1, BJHSP1, and BJHSPZ. Twenty micrograms of genomic DNA was loaded onto each lane, after digestion with restriction enzymes. Ra- diolabelled cDNA for each protein was then used to probe the DNA fragments. For each gene, the three lanes shown correspond to digestion with Bam HI, Eco RI, and Hind III, from left to right. The results suggest that each cDNA represents a single gene, and that the genes are not located in immediately proximal positions (within the resolution of the analysis). Arrows show the positions of positive signals. Molecular size scale in kilobase pairs is shown along left.

mediately proximate physical location of genes for any pair of the proteins.

DISCUSSION This study provides the first direct assessment of

transcription of JH-sensitive genes expressed during larva to adult differentiation. In connection with pre-

AJHSPi ARYLPHORIN

on day 2 (at lights on, and 12 hrs later). While all four proteins are present in the fat body on day 4, the basic proteins have been cleared from the hemolymph (BJHSP1, BJHSP2) while the two acidic proteins (arylphorin, AJHSP1) remain abundant in the hemolymph.

vious studies on the proteins examined here, a comprehensive understanding is now possible on levels of regulation of several JH-sensitive proteins.

Regulation of Gene Expression We have identified the rate of transcription as a level

of regulation of expression of a set of genes during metamorphosis, although the specificity of the regulation is not the same for all such genes. These data demonstrate JH effects on the 1) absolute magnitude of transcription, averaged across all genes; 2) absolute magnitude of transcription of specific metamorphosis- associated genes; and 3) specific regulatory effects on the transcription of genes analysed as a proportion of the total genomic transcription.

Maintenance of a high JH titer during what should normally be the final stadium (which normally has a low JH titer) caused an increase in overall transcription. Under normal conditions, the larvae feed for 2 days before experiencing a pulse of ecdysteroids that reprograms or curtails the expression of many genes [Riddiford, 19851. During those 2 days of feeding, the overall rate of transcription is already high. However, when the JH titer is maintained abnormally high during those 2 days, the magnitude of total transcription attained on the second day is distinctly higher than normal.

General hormonal effects. It has been observed in Lepidoptera that following the ecdysteroid pulse that terminates feeding and commits the final larval instar to pupation, total RNA synthesis sharply decreases [locke, 19701 and many more proteins decrease or cease expression than increase or begin expression [Riddiford, 1982; Kiely and Riddiford, 19851. These ob- servations complement the results obtained here in which total nuclear transcription decreased abruptly


Arylphorin

ARiSPl

BJHSPl BIHSP2

‘SUSTAINED HIGH JH TITER infcmd ngulatOV NCRMAL FINAL STADIUM inferred regulatory flow JH titer) mechanism during find stadium mechanism

1 2 3 4 1 2 3 4 + stability of m w -+ + +I transcript D3,D4 + :: :: ::+} decreased D3,D4

instability of transcription + + + mRNA abundance + + t

VanslaIablemRNA + + + abundance in FB + + + abundancein + + + hernolymph

transcription 2 +

translatablemRNA 2 + + abundance in FB i + abundancein 2 + hernolymph

transcription - + mRNA abundance - + + aanslatablcmRNA - + + abundance in FB - + abundancein - + hernolymph

mRNAabundance 2 + +

+ +

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+ + 1 or degradation D4 lack of clearance

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or degradation D4

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translatability of mRNA D4

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+ + +> uptake into FB D4 quantitative ] clearanceonD4

dvrrsbed stability of transcript D2-D4 untranslatibility ofmRNAD4

*hi$ JH titer of penultimate stadium maintained during final stadium by application of JH analog prior to molt to final instaq FB = fat bods symbols indicate relative activity or abundance as follows: - = not detected; 2 = limit of detectabilicy; one. trro or three plus symbols indicate increasingly higher levels of activity or abundance

F i g . 5. Regulatory levels at ,which the expression of the hemocyanin-related proteins is controlled in normal and hormone-treated final instar larvae. For each protein, five levels of regulation are presented, along with the relative activity at each level on days 1-4 of final instar larvae. Data for transcription and relative protein abundance

on day 3, the day that follows ecdysteroid-induced pupal commitment and cessation of feeding in T. ni [Jones et al., 19861. This general effect to decrease the transcription of many genes is modulated by juvenile hormone [Riddiford, 19851. A physiological basis for this phenomenon may be in the needs of “growth’ vs. “differentiation.” Prior to the general body commitment for the molt of the larva to a pupa, the larva is primarily concerned with increased larval size, which would require high rates of protein synthesis. However, once the larva is hormonally committed for differentiation to the pupa, its concern is not increase in size, but change in form. The latter process need not require the massive levels of protein synthesis that typify the larval growth period.

Specific hormonal effect. The present study has demonstrated hormonal effects on the transcription of specific genes above and beyond the general effects of J H on genomic transcription that are presumably mediated by generally-acting transcription factors. Under abnormal maintenance of high JH conditions during the final stadium, AJHSP1, BJHSPl, and BJHSPB all show a distinct suppression in their transcription on day 2. Since under these high J H conditions the rate of overall gene transcription is higher than normal on day 2, the relative proportion of total transcription due specifically to AJHSP1, BJHSPl, and BJHSPB on that

in the fat body (FB) and in the hernolymph are from the present paper. Data for mRNA abundance, translatable mFtNA, and protein synthesis are from Jones et al. (1990) and Jones et al. (1993). Where data permit, an inference is presented on the level or mechanism of regulatory action.

day is strongly suppressed by high JH. Another member of the hemocyanin superfamily, arylphorin, was not specifically affected by maintenance of a high JH titer. In fact, its rate of transcription increased on days 1 and 2 similarly in both normal and JH-treated larvae and was closely correlated with the overall rate of transcription averaged across the genome. These results strongly suggest that an important difference will be found in regulatory sequences between the arylphorin gene and the other three (JH-sensitive) members of the hemocyanin superfamily (AJHSP1, BJHSP1, and BJHSPB). Studies on other species of Lepidoptera have found that the biosynthesis of arylphorin and female- specific protein parallel changes in their mRNA abundance [Riddiford and Hice, 1985; Webb and Riddiford, 1988a,b; Kumaran et al., 1987; Izumi et al., 19881. Since some of those authors found that arylphorin mRNA does occur at a low level in preultimate instar larvae, it is important to determine if the increase in abundance of arylphorin mRNA at the final instar is due to an increase in transcription or a decrease in degradation.

Toward answering such questions, the results on transcription and abundance of expressed proteins presented here, in connection with our previous reports on mRNA abundance and translatability [Jones et al., 1993, 19901, enable identification of several mecha-

330 JONES ET AL. nisms of regulation of expression of these proteins, summarized in Figure 5. It is seen that while these proteins are all expressed during larval metamorphosis, different combinations of regulatory mechanisms are used for AJHSPl vs. arylphorin vs. BJHSPli BJHSPB.

Parameters of regulation of hemocyanin-related proteins in T. ni-sensitivity of transcription to JH. The first regulatory dichotomy that appears when comparing these proteins is the nonsensitivity of arylphorin transcription to high JH, in contrast to the sensitivity exhibited by the other three proteins. Those which are specifically transcriptionally suppressed by J H (AJHSP1, BJHSP1, BJHSPB) possess the common features of transcription and translation on day 3, which then markedly decreases (transcription) or become undetectable (translation) on day 4. In contrast, the arylphorin mRNA shows a distinctively sharp decline in abundance and in detectable translation by a full day earlier, although both its relative and absolute transcription rates paradoxically reach their highest relative levels on day 4 (Fig. 2).

Sensitivity to JH of stability of transcript. An- other difference in regulation between arylphorin and AJHSPl, BJHSPl, and BJHSPB is the distinctive difference in stability of their transcripts under normal and high-JH conditions, summarized in Figure 5. In normal larvae, the relative transcription of all the genes studied is high on day 3, and the values for all four of the genes are within severalfold of each other. Yet, while the relative abundance of the transcripts for AJHSP1, BJHSPl, and BJHSP2 are a t their highest relative abundance on day 3, the relative abundance of the transcript for arylphorin is so low on day 3 that it is essentially undetectable (over an order of magnitude lower by scanning densitometry). Thus, under normal conditions the undetectably low relative level of arylphorin transcript on day 3, despite high relative transcription rates of that gene, must be due to instability of the transcript.

However, if JH is abnormally maintained at a high level during the final instar feeding stage, the stability of the transcripts for the three transcriptionally sensitive proteins, and especially BJHSPl and BJHSPB, is distinctly reduced below normal. The result is that while transcription is about half-normal under high JH conditions, northern analysis reveals no detectable ac- cumulating transcripts on days 1-3. In contrast, the arylphorin transcript does not exhibit such symptoms of high turnover under such conditions (Fig. 5). Studies on expression of cuticle genes in M. sexta have sug- gested that the stability of some ecdysteroid-sensitive transcripts is reduced by ecdysteroids [Hiruma et al., 19911. At this point, it cannot be determined whether JH and ecdysteroids affect message stability by a similar mechanism. We also note that we have inferred stability from transcription rates and transcript abundances and that calculations of apparent stability is

based on the premise that isolated nuclei faithfully re- produce in vivo transcription rates.

Differential protein uptake by fat body. In most Lepidoptera, the fat body is the major biosynthetic source of the storage protein found in the hemolymph and is also the site of subsequent storage of some of these proteins [Kanost et al., 1990; Palli and Locke, 1987a,bl. In T. ni, an additional dichotomy in regulatory mechanisms concerns clearance of the proteins from the hemolymph (Fig. 5). Under normal conditions, the two basic proteins are quantitatively cleared from the hemolymph on day 4 (when their transcript and translation are undetectable), with considerable uptake by the fat body. In contrast, while some AJHSPl (JH sensitive) and some arylphorin (not JH sensitive) is sequestered into the fat body on day 4 (when their transcript and translation are undetectable), the titer of these proteins in the hemolymph on day 4 is not greatly different than on day 3. This partial reuptake by the fat body appears to be induced by the second, and higher, of the two final instar ecdysteroid peaks, the latter of which occurs late on day 3 in T. nz [Jones et al., 1986; Miller and Silhacek, 1982; Tojo et al., 1978; Caglayan and Gilbert, 19871.

Sensitivity to JH of translatability of the mRNA. The two basic proteins also appear to differ from AJHSPl and the arylphorin in the effects of maintained high JH on translatability of the message (Fig. 5). When the feeding stage of the final instar is ex- tended from 2 days to 4 days by exogenous application of the JH analog, mRNA for the two basic proteins becomes easily detectable on day 4, but not sooner. However, these transcripts present on day 4 do not translate in a rabbit reticulocyte lysate system [Jones et al., 19931. In contrast, the mRNAs from the corresponding treatment for AJHSPl are translatable. Since these mRNAs are purified away from cellular proteins prior to in vitro translation, the mechanism of inhibited translation must lie with a physical property of the message itself [Sekeris and Scheller, 19771.

Relationships Among the Genes Studied Previous studies have documented that AJHSP1,

BJHSPl, and BJHSPB are members of the hemocyanin superfamily [Telfer et al., 1983; Jones et al., 1990, 19931, a group which also contains members identified as arylphorin [Ryan et al., 1985; Fujii et al., 19891 and met-rich/female-specific protein [Sakurai et al., 19881. The results of genomic Southern blotting showed that the genes for arylphorin, AJHSPl, BJHSP1, and BJHSPB in T. ni are apparently single copy genes. Some moth chorion protein genes are organized as pairs of genes in opposite orientation, each pair sharing a common promoter [Jones and Kafatos, 19801. At the level of resolution used here, the results do not suggest the genes for these proteins are so tightly linked or have an organization such as found in chorion genes.


CONCLUSIONS The different groups of genes studied here (basic,

acidic, and arylphorin protein genes) are each regulated by several distinct combinations of parameters that govern their expression and final protein abundance. Certain subgroups of genes possess certain regulatory features in common. The genes which are sensitive to JH are governed by mechanisms operating at the levels of transcription, mRNA stability, and translation. These related proteins with transcriptional sensitivity, or insensitivity, to J H apparently do not phys- ically share the same promoter, which strongly suggests that comparison of these independent promot- ers for conserved sequences will reveal elements through which regulation by JH is mediated. The results presented here also predict the existence of molecules affecting message stability whose appearance or activity is influenced by JH. Since the basis for regulation of transcript stability by the related vertebrate hormone retinoic acid [Gellersen et al., 1992; Antras et al., 19911 is not well understood [Nielsen and Shapiro, 19901 the utility of this insect system for analysis of such JH-effects should reveal mechanisms of transcript regulation that are of general interest.

ACKNOWLEDGMENTS We thank Dr. Davy Jones for his helpful suggestion

during the study. This research was supported in part by NIH grant DK 39197, NSF grant 9005184, and a Biomedical Research Support Grant.

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