Neuron,

Vol. 5, 411-419,

October,

1990, Copyright

0 1990 by Cell Press

Activation of Polyubiquitin Gene Expression during Developmentally Programmed Cell Death Lawrence M. Schwartz, Anita Myer, Lucy Marcy Engelstein, and Carolina Maier Department of Zoology Merrill Science Center University of Massachusetts Amherst, Massachusetts 01003

Kosz,

Summary Ubiquitin, a highly conserved 76 amino acid protein, plays a role in targeting intracellular proteins for degradation. Ubiquitin expression was examined during the developmentally programmed atrophy and degeneration of the intersegmental muscles (ISMs) in the hawkmoth, Manduca sexta. A clone containing nine repeats of the ubiquitin coding sequence was isolated from an ISM cDNA library and was used as a probe to examine polyubiquitin expression during development. When the ISMs became committed to degenerate, polyubiquitin gene expression increased dramatically. Injection of 2@hydroxyecdysone, which delays degeneration in this system, prevented the increase in polyubiquitin mRNA. The expression of polyubiquitin occurred without apparent activation of the cell’s heat shock response. These data suggest that ubiquitin plays a role in programmed cell death. introduction Ubiquitin isa highlyconserved, 76aminoacid protein found in all eukaryotic cells (Schlesinger et al., 1975). When covalently linked to proteins as a posttranslational modification, ubiquitin often targets the protein to the ATP-dependent proteolysis system in cells (Hershko, 1988; Mayer et al., 1989). There are several ubiquitin genes present in the genome, one of which encodes multiple head-to-tail repeats of the ubiquitin protein (Ozkaynak et al., 1987). This polyubiquitin gene is activated during metabolic distress associated with the heat shock response (Finley et al., 1987); in this response it probably participates in the removal of denatured proteins from the cytoplasm. Recently, ubiquitin has been found to be a component of the cytoplasmic inclusions of Alzheimer’s disease and several other degenerative disorders (Mori et al., 1987; Manetto et al., 1988; Gallo and Anderton, 1989), although its role in this pathological cell death is currently unknown. Cell death serves a variety of functions in the normal development of animals (Bowen and Lockshin, 1981). Much of the sculpting of the body form involves the removal of select cells, such as can be seen during the formation of the digits during embryogenesis (Hinchliffe, 1981). Cell death can also serve to adjust the number of neurons innervating central or peripheral targets (Hamburger and Oppenheim, 1982). It

can also remove obsolete tissues during development. For example, during metamorphosis in both amphibia (Weber, 1965) and insects (Finlayson, 1956; Schwartz and Truman, 1984; Kimura and Truman, 1990), many embryonically derived tissues are degraded during the formation of the adult animal. One system that has been particularly useful for examining the biochemical events mediating programmed cell death are the intersegmental muscles (ISMs) of the tobacco hawkmoth, Manduca sexta. These giant, embryonically derived fibers are responsible for the locomotory behaviors of the larva and the defensive and emergence behaviors of the developing adult. On day 15 of pupal-adult development, the ISMs begin to atrophy as a result of a decline in the circulating titer of the insect molting hormone, 20hydroxyecdysone (20-HE) (Schwartz and Truman, 1983). Atrophy involves the loss of muscle mass, without change in either morphology or physiological function (Schwartz and Truman, 1984; Schwartz, unpublished data). The ISMs continue to atrophy until the day of adult eclosion (emergence) from the pupal cuticle (day 18). A further decline in ecdysteroids early on day 18 commits the ISMs to degenerate (die) during the 36 hr following adult eclosion. If animals are treated with 20-HE on day 17 of development, degeneration of the ISM is delayed until the steroid titer subsequently falls. Several lines of evidence suggest that the commitment of the ISMs to degenerate involves specific changes in gene expression (Schwartz and Kay, 1987, Sot. Neurosci., abstract; Schwartz et al., 1990). We have examined the expression of ubiquitin during ISM development to determine whether it represents one of these cell death genes. This report demonstrates that the polyubiquitin gene is selectively expressed during muscle atrophy and degeneration, is under hormonal control, is expressed independently of the cell’s heat shock response, and serves as an early molecular marker for a cell’s commitment to die.

Results Characterization of the Polyubiquitin cDNA To study ubiquitin expression during programmed cell death in Manduca, a cDNA librarywas constructed in Lambda Zap II (see Experimental Procedures) with poly(A)+ RNA isolated from day 18 (committed) ISMs. The library had 584,000 original recombinants with an average insert size of 1.6 kb. To isolate polyubiquitin sequences from Manduca, the library was screened with a Drosophila polyubiquitin gene (Lee et al., 1988). Several clones were identified and plaque-purified, and the cDNA inserts were isolated within the Bluescript vector (Stratagene) via in vivo excision. One of these clones, 1’1, appeared to contain a full-

5’

15'

30'

45'

60’

bP R

l636-

1018-

506-

I3

Figure 2. Determination of the Number Encoded by a Polyubiquitin cDNA

Monomers

The Bluescript plasmid containing clone 1’1 was partially digested with Bglll, fractionated in 1.2% agarose, and transferred to a Zeta-Probe nylon membrane. (A) The blot was probed with a 3ZP-labeled cbiquitin clone, MstJb, which contains Manduca ubiquitin coding region. The sizes of the resulting polymers agrees well with the 228 bp ubiquitin monomer repeat. The first and last ubiquitin repeats (see [B]) remain on the piasmid. (8) Model for the organization of the cDNA clone 1’1. The nine boxes represent the head-to-tail repeats of the ubiquitin coding region; the bold lines represent the untranslated regions of the clone.

ct5 Figure 1. Nucleotide and Predicted Ammo Acid the 5’and 3’Ends of the Clone 1’1, Which Encodes ubiquitin The initial methionine the terminal asparagine sites are underlined.

of Ubiquitin

Sequences Manduca

for Poly-

ai the start of each ubiquitin repeat and are marked in bold; the Bglll restriction

length recombinant. Figure 1 shows both the nucleotide and predicted amino acid sequences for a portion of clone 1’1: At the 5’ end of this region of the clone, there were 46 bases of untranslated sequence, followed by one complete 228 bp ubiquitin monomer and a portion of a second one. At the 3’ end of the clone, another full monomer and a partial monomer were sequenced, along with a 132 bp untranslated region. The sequenced ubiquitins all encoded identical proteins, except for the C-terminal repeat, which had an additional terminal amino acid, asparagine. The ubiquitin proteins from Manduca, Drosophila, and human share 100% identity (Lee et al., 1988; Wiborg et al., 1985). At the DNA level in Manduca, there was considerable sequence variation among the various ubiquitin coding regions, primarily at the third base. Each of the ubiquitin repeats, except for the 5’-most, had a unique Bglll restriction site at the beginning of the coding region. Rather than sequence clone 1’1 in its entirety, we took advantage of the repeated Bglll restriction sites at the 5’ end of the ubiquitin monomers. 1’1 was partially digested with Bglli, fractionated in agarose, blot-

ted, and probed with 32P-labeled ubiquitin DNA. As can be seen in Figure 2A, a ladder composed of up to seven ubiquitin monomers was obtained. The molecular weights of the rungs agreed well with multiples of the 228 bp size determined from DNA sequencing of ubiquitin. Two of the ubiquitin monomers are not seen in the gel, since the 5’ most repeat lacks the Bgili site and the 3’ most ubiquitin remains on the vector (data not shown). Taken together, the sequencing and digestion data suggest that 1’1 encodes nine head-totail repeats of the ubiqoitin coding sequence (Figure 2B). Analysis of Ubiquitin Expression Using 1’1 as a probe, we examined the pattern of polyubiquitin gene expression during muscle development. The ISMs were removed from animals at various stages of pupal-adult development, starting before the onset of atrophy (day 14) and extending well into the degeneration program (day 18, 5 hr posteclosion; 5 hr P. E.). As a control, one group of insects was injected on day 17 of development miith 25 j.rg of 2Q-HE, since this dramatically delays the time of ISM degeneration (Schwartz and Truman, 1983). The lSMs from these animals were removed 5 hr after the normal time of eclosion on day 18. RNA was isolated from the ISMs, fractionated by size, transferred to a nylon membrane, and hybridized with 32P-labeled 1’1 DNA. The probe hybridized to

Polyubtqurtin

Expressron

during

Cell Death

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123456

A d14

d15

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d17

d18

3hr P.E.

5hr P.E.

5hr + 20-HE 4 +

Ftgure 4. Levels Manduca

mRNA

tn Different

Tissues

of

Several tissues were isolated from day 18 Manduca, and the RNA was isolated. Total RNA (15 pg) was fractionated, blotted, and probed wrth l?P-labeled 1’1 DNA. Lane 1, Malpighian tubule; lane 2, fat body; lane 3, flight muscle; lane4, intersegmental muscle; lane 5, male sexual accessory gland; lane 6, ovary and oocytes.

6

d14

of Polyubiquitin

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d16

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d18

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+

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Figure 3. Developmental with 1’1 and 18-2 cDNAs

Northern

Blots

of ISM

RNA

Probed

(A) Total RNA was isolated from the ISMs at the developmental stages indicated. d, day of development; PE., hours posteclosion (on day 18 of development). Total RNA (15 ug) was fractionated under denaturing conditions in agarose, transferred to a mem1’1. The arrowheads brane, and hybridized to clone 3zP-labeled identify three hybridizing transcripts of 4.3, 2.6, and 1.7 kb. (B) A blot comparable to that in (Alwas probed with clone 18-2, which encodes a ubiquitin fusion protein.

several developmentally regulated transcripts (Figure 3A). The transcripts ranged in size from 4.3 to 1.7 kb (Figure 3A, see arrowheads). The intermediate sized transcript, 2.6 kb, is approximately the same size as the insert for clone 1’1. These multiple transcripts may be the products of either alternate splicing or distinct genes. We have isolated two different Manduca polyubiquitin genomic clones, which suggests that the transcripts are from distinct genes (Myer and Schwartz, unpublished data). The abundance of polyubiquitin transcripts changed dramatically during development. Levels of polyubiquitin mRNA were low on day 14, but accumulated early in the atrophy phase (days 15 and 16), when the ISMs lose mass but are physiologically normal (Schwartz and Truman 1984; Schwartz, unpublished data). Transcript levels dropped on day 17 and then dramatically increased when the ISMs became committed to die (day 18 and posteclosion). In fact, early on day 18, the ISM are committed to die, but have not yet displayed any of the morphological or physiologi-

cal changes that accompany cell death (Schwartz, unpublished data). When animals were treated on day 17 with 20-HE and their ISMs examined 5 hr after the eclosion of control animals, the levels of polyubiquitin mRNA were greatly reduced. This pattern of expression correlates well with the atrophy and degeneration programs displayed by the ISMs (Schwartz and Truman, 1983). All eukaryotes examined thus far have additional ubiquitin genes that encode a single copy of the ubiquitin protein fused in frame at the C-terminus to another protein (Ozkaynak et al., 1987). We have previously cloned and sequenced one of these ubiquitin fusion genes from the ISMs, clone 18-2 (Schwartz et al., 1990; Bishoff and Schwartz, submitted). We wanted to ensure that the transcripts identified in Figure 3A were products of the polyubiquitin gene and not other ubiquitin-containing sequences. A Northern blot, identical to the one used in Figure 3A, was probed with radiolabeled DNA from clone 18-2. As can be seen in Figure 3B, this probe labeled the developmentally regulated transcripts seen above, as well as an abundant constitutively expressed RNA. The constitutive sequence is the ubiquitin fusion gene product (Bishoff and Schwartz, submitted). This blot shows that there is equal loading of RNA in each lane and that not all ubiquitin genes are developmentally regulated or influenced by exposure to 20-HE. To ensure that the expression of polyubiquitin in day 18 ISMs was correlated with the commitment to degenerate and not just the developmental age of the animal, different tissues were surveyed for their polyubiquitin mRNA content. Six day 18 preeclosion animals were dissected, and the following tissues were isolated for RNA extraction: fat body, flight muscle, ISM, ovary, male sexual accessory gland, and Malpighian tubule. As seen in the Northern blot in Figure 4, all tissues expressed polyubiquitin, as has been noted in other species (Lee et. al., 1988). However, the greatest hybridization signal was seen in the ISMs. Since the levels of ubiquitin mRNA increased dramatically coincident with the commitment of the

d14

d15

d16

d17

d18

3hr P.E.

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5hr P.E.

5hr 2Q-HE

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9768B

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D14

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Df8

D17

D18

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14hr P.E.

18Figure

5. Ublquitin

Western

Blot of ISM Proteins

Equal amounts of ISM protein from the stages indicated were separated on a 10% SDS polyacrylamide gel and transferred to a membrane. The blot was reacted with a rabbit polycional antibody against conjugated ubiquitin (Haas and Bright, 1985). A goat anti-rabbit secondary antibody, labeled with horseradish peroxidase, was used to detect the primary antibody.

ISMs to degenerate, we examined the pattern of ubiquitin protein expression. ISM were removed from animals at various developmental stages, and equal amounts of protein were fractionated on polyacrylamide gels. After transfer to a membrane, the proteins were reacted with an affinity-purified rabbit polyclonal antiserum directed against conjugated ubiquitin (Haas and Bright, 1985). Since the majority of the ubiquitin in cells is conjugated to other proteins, the antibody labeled a large number of proteins on Western blots. As can be seen in Figure 5, ubiquitin conjugates were present in the ISMs at all stages of development. In some cases, major contractile proteins such as actin (43 kd) appeared to be labeled, as has been seen in other insect muscle (Ball et al., 1987). At the time that the muscles become committed to degenerate on day 18, ubiquitin conjugation appeared to increase. Once the degeneration process began, ubiquitination increased dramatically and new ubiquitin-containing bands appeared on the blot. In separate experiments, it has been shown that ubiquitin conjugation increases IO-fold on day 18 (Haas and Schwartz, unpublished data). Expression of Heat Shock 70 The polyubiquitin gene is activated during metabolic stress in many cells and is considered to be one of the heat shock genes (Finley et al., 1987). It is possible that the metabolic changes associated with degeneration activates the heat shock response in the ISMs. If this were true, then the expression of polyubiquitin might be secondary to the cell death process, rather than a

Figure within

6. Developmental the ISMs

Changes

in hsp70

RNA

and

Protein

(A) Total RNA was isolated from the ISMs at the developmental stages indicated. Total RNA (15 pig) was fractionated under denaturing conditions in agarose, blotted, and hybridized to the BamHI-EcoRI fragment of the pDM300 cione encoding Drosophila hsp70 (McCarry and Linquist, 1985). d, day of development; PE., hours posteclosion (on day 18 of development). (B) HSP70 Western blot of ISM proteins at different developmental stages. Equal amounts of ISM protein from the stages indicated were separated on a 10% SDS polyacryiamide gel and transferred to a membrane. The blot was reacted with a mouse monoclonal antibody directed against the Drosophila HSP70 protein. An alkaline phosphatase secondary antibody was used to detect the antigen.

possible participant in it. To determine whether the ISMs were activating the stress response, Northern blots were probed with radiolabeled DNA from the Drosophila hsp70 gene (McGarry and Linquist, 1985) (Figure 6A). This blot displays an abundant transcript that was present at every stage of development. Since there was some unevenness in quantity of the transcript, we reprobed the blot with a ubiquitin fusion cDNA (clone 18-2), which is constitutively expressed at high level in the ISMs (see Figure 38). The abundance of the 18-2 transcript paralleled the amount of hsp70 RNA on the blot, supporting the observation that hsp70 is consitutively expressed throughout ISM development (data not shown). When these blots are probed with different regions of the Drosophila hsp70 cDNA, a second hybridizing band can be visualized, which presumably encodes a cognate gene product (Kosz and Schwartz, unpublished data). The levels of HSP70 protein were also examined during ISM development. ISM proteins were fractionated and blotted as described above for Figure 5. The blot was reacted with a mouse monoclonal antibody directed against the Drosophila HSP70 protein (Kurtz et al., 1986). This blot displays two distinct constitutively expressed bands, which presumably reflect HSP70 and a cognate (Figure 68).

Polyubiquitin

Exprewon

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Cell Death

415

Figure

7. Patterns

of Ubiquitin

mRNA

and

Protein

Accumulation

Sections (IO urn thick) were examined from day 17 (A and C) and day 18 (Band D) ISMs. In situ hybridization DNA probe to polyubiquitin was performed on single ISM fibers (A and B). Several ISM fibers were stained directed against ubiquitin (C and D). Bar -130 pm (A and B); 87 urn (C and D).

Morphological

Examination

of Ubiquitin

The ting

described to demonstrate

above

experiments methods

Expression

utilize various that polyubiquitin

blotis

expressed generate. sion, we

when the ISMs To determine the used the techniques

with a digoxigenin-labeled with a polyclonal antibody

become committed to decellular patterns of expresof in situ hybridization

Nt?UKX 416

Figure 8. Ubiquitin Stalntng oi Neurons

lmmunocytochemlcai

The fourth abdominal ganglion was taken from a moth 16 hr after eclos~on and examrned for ublquitln staining. Two motor neurons are present, one of which stains IF tensely with an anti-ublquitin polyclonal antibody.

and immunocytochemistry to study the ISMs. The ISMs were removed from the animals before and after the time of commitment (days 77 and 18) and prepared for examination (see Experimental Procedures). As suggested by the various blots above, there is a dramatic increase in polyubiquitin mRNA on day 18 relative to day 17. To demonstrate this histologically, DNA from clone 1’1 was labeled with digoxigenin and used as a hybridization probe on cryostat cut sections from the ISMs on days 17 and 18. On day 17, only sparse hybridization signals were detectable in the ISM (Figure 7A). In contrast, the day 18 ISM section displayed intense patches of label, reflecting the increase of ubiquitin mRNA abundance (Figure 7B). At the protein level, ubiquitin, presumably conjugated to contractile proteins, was uniformly present at low levels in day 17 ISMs (Figures 7C). On day 18, prior to eclosion, staining for ubiquitin was much more intense (Figure 7D). Interestingly, ubiquitin staining was not uniform across the fiber, but instead appeared to be concentrated over the contractile proteins away from the sarcolemma. The muscles used in these experiments were taken from animals prior to eclosion

or

the

onset

of

degeneration.

Therefore,

ubiquitin was a strong indicator of the cell’s commitment to degenerate. Large numbers of motor and interneurons are also lost following adult eclosion in Manduca (Truman and Schwartz, 1984). The trigger for neuron degeneration is the same as that for the ISM: a fall in the circulating ecdysteroid titer. To determine whether the same biochemical pathway used in the muscle might be used in the nervous system, we examined motor neurons. The fourth abdominal ganglia from animals 16 hr after eclosion were fixed, sectioned, and stained with the anti-ubiquitin antibody. Figure 8 shows two motor neurons that displaycytoplasmic staining with the antibody. One of the two motor neurons is more intensely labeled with the antibody, suggesting the accumulation of ubiquitin. While we do not yet know whether this is one of the identifiabie motor neurons that will die following eclosion, these preliminary data suggest that ubiquitin is differentially expressed at the time when many neurons will die. We are currently performing a systematic examination of ubiquitin staining in motor neurons from various stages of development (Truman and Schwartz, unpublished data).

Polyubiquitin 417

Expression

during

Cell Death

Discussion The ISMs of Manduca have proven to be a valuable system for the study of programmed cell death. The muscles become committed to die as a result of a decline in the ecdysteroid titer, which in turn leads to the activation of specific sets of genes (Schwartz and Truman, 1983; Schwartz et al., 1990). We have cloned a number of ecdysteroid-regulated genes from the ISMs that appear to participate in the cell death process. In the present study, we report that one of these genes encodes polyubiquitin. The Manduca polyubiquitin gene, like that of other species, encodes multiple head-to-tail repeats of the ubiquitin protein. In the case of clone 1’1, there are nine copies of the monomer. This provides a powerful amplification system, since the translation of each copy of the mRNA produces almost an order of magnitude increase in the number of free ubiquitin proteins. Cells maintain a balance between the free and conjugated ubiquitin within the cytoplasm (Haas and Bright, 1987). In the ISMs, the increase in free ubiquitin on day 18 causes a IO-fold increase in the number of ubiquitin-conjugated proteins (Haas and Schwartz, unpublished data). This agrees well with the changes in ubiquitin pools seen in atrophying rat muscle (Haas, 1988). Presumably, the increase in ubiquitinization increases the rate of protein degradation required for cell death. Morphological experiments presented here show that ubiquitin mRNA and protein accumulate in the ISMs. In addition, ubiquitin appears to accumulate in certain motor neurons at a time when large numbers of the motor neurons become committed to degenerate in both Manduca and Drosophila (Truman and Schwartz, unpublished data). Since the accumulation of ubiquitin precedes any of the morphological or physiological changes that accompany cell death, it can serve as an early molecular marker for the cell’s commitment to die during development. In mammals, other genes that are abundantly expressed at the time of cell death have been identified. Much of this work has been performed with the rat ventral prostate, in which castration induces tissue regression. Castration-induced atrophy results in the expression of several genes within the prostate, including testosterone-repressed message 2 (Monpetit et al., 1986), p-transforming growth factor (Kyprianou and Isaacs, 1989), c-fos, and 4170 (Buttyan et al., 1988). Since hsp70 expression is not altered during ISM degeneration, castration-induced prostate regression may involve processes different from those involved in programmed cell death. At present, polyubiquitin expression has not been examined during either prostate regression or vertebrate embryonic development. In humans, ubiquitin has been shown to accumulate in the cytoplasmic inclusions of several neurodegenerative diseases (Callo and Anderton, 1989). A variety of methods have been used to demonstrate that ubiquitin is a component of the inclusion bodies

in such disorders as Alzheimer’s disease (Mori et al., 1987), Picks disease, motor neuron disease (Leigh et al., 1988), and Parkinson’s disease (Manetto et al., 1988). At present, the role of ubiquitin in the pathogenesis of these diseases is unknown. Presumably, early cellular pathology produces denatured or damaged proteins that then become ubiquitinated to mediate their removal from the cytoplasm. Given that ubiquitin appears to participate in developmentally programmed cell death as well, these observations support the notion that ubiquitin is a common element in cell degeneration. Experimental

Procedures

Animals The tobacco hawkmoth, M. sexta, was reared as previously described (Bell and Joachim, 1976). Comparably sized animals were staged during development according to specific cuticular markers (Schwartz and Truman, 1983). For animals treated with ZO-HE (Sigma), a Hamilton syringe was used to introduce approximately 25 PI volumes into the dorsal thorax. RNA The ISMs have high levels of RNAase (Schwartz, unpublished data), and so great care was required in isolating RNA. Animals were rapidly dissected under ice-cold saline (Schwartz and Truman, 1983), and the ISMs were frozen in liquid nitrogen. Muscles from IO-20 staged insects were homogenized with a Tissuemizer (Tekmar Co.) in 4 M guanidinium isothiocyanate extraction buffer according to MacDonald et al. (1987). Total RNA was isolated by centrifugation through a 5.7 M CsCl pad. The pellet was resuspended in 5% B-mercaptoethanol, 0.5% Sarkosyl, 5 mM EDTA, phenol-chloroform extracted, and ethanol-percipitated. Poly(A)+ RNA was isolated by oligo(dT)-cellulose chromatography (Jacobson, 1987). For Northern blots, 15 ug of total RNA was denatured in formaldehyde and separated by size in 1.5% agarose (Fourney et al., 198&T), prior to transfer to Zeta-Probe membranes (Bio-Rad). Blots were hybridized overnight at 65OC under stringent conditions in 1 mM EDTA, 7% SDS, 0.5 M NaH2P04 (pH Z2). The 32P-nicktranslated DNA was used as a probe (described below). Blots were autoradiographed with Kodak XAR film. cDNA library Construction and Screening cDNA was synthesized from day 18 ISM polyA+ RNA with a cDNA synthesis kit (Pharmacia LKB Biotechnology), ligated to EcoRl linkers, and cloned into Lambda Zapll (Stratagene). The library had approximately 584,000 original recombinants, with an average insert size of 1.6 kb, and was amplified once before use. The library was plated out at high density, and duplicate filter lifts were obtained (MS]). The filters were probed with the 32P-nick-translated, 6.2 kb Hindlll-Hindlll fragment from the Drosophila polyubiquitin genomic clone pUB3 (Lee et al., 1988). This fragment contains ILLcopies of the ubiquitin coding region, along with some 5’ and 3’ noncoding sequence. Hybridizing recombinants were plaque-purified, and the cDNA clones were recovered within the Bluescript vector (Stratagene) by in viva excision. Of the several clones isolated, one was selected for analysis, clone 1’1. DNA was sequenced with Sequenase (US Biochemicals) by the dideoxy method of Sanger et al. (1977). Western Blots ISMs were homogenized in Laemmli extraction buffer and fractionated on 10% SDS polyacrylamide gels (Laemmli, 1970). Proteins were then electroblotted to Immobilon-P membranes (Millipore) and reacted either with an affinity-purified rabbit polyclonal antibody against protein-conjugated ubiquitin (Haas and Bright, 1985) or with a mouse monoclonal antibody against Drosophila HSP70 (Kurtz et al., 1986). Primary antibodies were

Neuron 418

visualized peroxidase

with secondary antibodies (ubiquitin blot) or alkaline

labeled with horseradish phosphatase (HSP70 blot).

Morphology For immunocytochemistry of the ISMs, animals were dissected under saline and the ISMs were pinned at length. The muscles were then fixed in 4% paraformaldehyde in buffer and embedded in paraffin according to standard protocols (Humason, 1972). To ensure equal treatment, 10 urn sections from both days 17 and 18 were placed on the same slides. Tissues were reacted with both a rabbit anti-ubiquitin antibody (Muller et al., 1988) and a horseradish peroxidase avidin-biotin detection kit (Vector). To examine the nervous system, the ventral nerve cord from 16 hr posteclosion was fixed and examined in the same manner as the ISMs. For in situ hybridization, ISMs from days 17 and 18 were pinned at length and frozen in liquid nitrogen. Tissues were sectioned (IO urn) in a cryostat and hybridized to a digoxigeninlabeled 1’1 probe (Boehringer Mannheim). The probe was detected with a alkaline phosphatase-labeled, anti-digoxigenin antibody, according to the manufacturer’s protocol.

Haas, A. L., and Bright, tion and quantitation jugates. J. Biol. Chem.

P. M. (1985). The immunochemical of intracellular ubiquitin-protein 260, 12464-12473.

Haas, A. L., and Bright, pools within cultured 262, 345-351.

P. M. (1987). The dynamics of ubiquitin human lung fibroblasts. J. Biol. Chem.

Hamburger, V., and Oppenheim, ring neuronal death in vertebrates.

deieccon-

R. W. (1982). Naturally Neurosci. Commun.

Hershko, A. (1988). Ubiquitin-mediated Biol. Chem. 263, 15237-15240.

protein

occur7,39-55.

degradation.

J.

Hinchliffe, J. R. (1981). Cell death in embryogenesis. In Ceil Death in Biology and Pathology, I. D. Bowen and R. A. Lockshin, eds. (New York: Chapman and Hall), pp. 35-78. Humason, G. L. (1972). Animal cisco: W. H. Freeman and Co.). Jacobson, A. (1987). RNA. Meth. Enzymol.

Tissue

Purification and 752, 254-261.

Techniques

(San

fractionation

Fran-

of poly(A)+

Kimura, K., and Truman, J. W. (1990). Postmetamorphic in the nervous and muscular systems of Drosophila ter. J. Neurosci. 70, 403-411.

ceil death melanogas-

Kurtz, S., Rossi, J., Petko, L., and Lindquist, S. (1986). An ancient developmental induction: heat-shock proteins induced in sporulation and oogenesis. Science 237, 1154-1157.

Acknowledgments We thank the following people for their generous provision of reagents: J. Buckner for Manduca eggs, S. Linquist for the clone and antibody for Drosophila HSP70, A. Haas for the provision of the ubiquitin antibody used on the Western blot, Sylviane Muller for the ubiquitin antibody used for histology, and J. Lis for the Drosophila ubiquitin pUB#3 clone. We also thank S. Robinson, R. Phillis, and R. Murphey for a critical reading of the manuscript, A. Haas for helpful discussions, and S. Bishoff for technical assistance. This work was supported by National Institutes of Health Grant GM40458 to L. M. S. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemenf” in accordance with 18 USC Section 1734 solely to indicate this fact.

Kyprianou, N., and Isaacs, J. T. (1989). Expression of transforming growth factor-8 in the rat ventral prostate during castrationinduced programmed cell death. Mol. Endocrinol. 3, 1515-1522.

Received

Manetto, V., Perry, G., Tabaton, M., Mulvihill, P., Fried, V., Smith, H. T, Gambetti, P., and Autilio-Cambetti, L. (1988). Ubiquitin is associated with abnormal cytoplasmic filaments characteristic of neurodegenerative diseases. Proc. Natl. Acad. Sci. USA 85, 4501-4505.

May

29, 1990;

revised

July 19, 1990.

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Activation of polyubiquitin gene expression during developmentally programmed cell death.

Ubiquitin, a highly conserved 76 amino acid protein, plays a role in targeting intracellular proteins for degradation. Ubiquitin expression was examin...
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