Polyubiquitin mRNA Research Articles

Metabolic rate and rates of protein turnover in food-deprived cuttlefish, Sepia officinalis (Linnaeus 1758).

To determine the metabolic response to food deprivation, cuttlefish (Sepia officinalis) juveniles were either fed, fasted (3 to 5 days food deprivation), or starved (12 days food deprivation). Fasting resulted in a decrease in triglyceride levels in the digestive gland, and after 12 days, these lipid reserves were essentially depleted. Oxygen consumption was decreased to 53% and NH4 excretion to 36% of the fed group following 3-5 days of food deprivation. Oxygen consumption remained low in the starved group, but NH4 excretion returned to the level recorded for fed animals during starvation. The fractional rate of protein synthesis of fasting animals decreased to 25% in both mantle and gill compared with fed animals and remained low in the mantle with the onset of starvation. In gill, however, protein synthesis rate increased to a level that was 45% of the fed group during starvation. In mantle, starvation led to an increase in cathepsin A-, B-, H-, and L-like enzyme activity and a 2.3-fold increase in polyubiquitin mRNA that suggested an increase in ubiquitin-proteasome activity. In gill, there was a transient increase in the polyubiquitin transcript levels in the transition from fed through fasted to the starved state and cathepsin A-, B-, H-, and L-like activity was lower in starved compared with fed animals. The response in gill appears more complex, as they better maintain rates of protein synthesis and show no evidence of enhanced protein breakdown through recognized catabolic processes.

Mechanisms of protein degradation in mantle muscle and proposed gill remodeling in starvedSepia officinalis

Cephalopods have relatively high rates of protein synthesis compared to rates of protein degradation, along with minimal carbohydrate and lipid reserves. During food deprivation on board protein is catabolized as a metabolic fuel. The aim of the current study was to assess whether biochemical indices of protein synthesis and proteolytic mechanisms were altered in cuttlefish, Sepia officinalis, starved for 7 days. In mantle muscle, food deprivation is associated with a decrease in protein synthesis, as indicated by a decrease in the total RNA level and dephosphorylation of key signaling molecules, such as the eukaryote binding protein, 4E-BP1 (regulator of translation) and Akt. The ubiquitination-proteasome system (UPS) is activated as shown by an increase in the levels of proteasome β-subunit mRNA, polyubiquitinated protein, and polyubiquitin mRNA. As well, cathepsin activity levels are increased, suggesting increased proteolysis through the lysosomal pathway. Together, these mechanisms could supply amino acids as metabolic fuels. In gill, the situation is quite different. It appears that during the first stages of starvation, both protein synthesis and protein degradation are enhanced in gill. This is based upon increased phosphorylation of 4E-BP1 and enhanced levels of UPS indicators, especially 20S proteasome activity and polyubiquitin mRNA. It is proposed that an increased protein turnover is related to gill remodeling perhaps to retain essential hemolymph-borne compounds.

Redox regulation of diaphragm proteolysis during mechanical ventilation

Prevention of oxidative stress via antioxidants attenuates diaphragm myofiber atrophy associated with mechanical ventilation (MV). However, the specific redox-sensitive mechanisms responsible for this remain unknown. We tested the hypothesis that regulation of skeletal muscle proteolytic activity is a critical site of redox action during MV. Sprague-Dawley rats were assigned to five experimental groups: 1) control, 2) 6 h of MV, 3) 6 h of MV with infusion of the antioxidant Trolox, 4) 18 h of MV, and 5) 18 h of MV with Trolox. Trolox did not attenuate MV-induced increases in diaphragmatic levels of ubiquitin-protein conjugation, polyubiquitin mRNA, and gene expression of proteasomal subunits (20S proteasome alpha-subunit 7, 14-kDa E2, and proteasome-activating complex PA28). However, Trolox reduced both chymotrypsin-like and peptidylglutamyl peptide hydrolyzing (PGPH)-like 20S proteasome activities in the diaphragm after 18 h of MV. In addition, Trolox rescued diaphragm myofilament protein concentration (mug/mg muscle) and the percentage of easily releasable myofilament protein independent of alterations in ribosomal capacity for protein synthesis. In summary, these data are consistent with the notion that the protective effect of antioxidants on the diaphragm during MV is due, at least in part, to decreasing myofilament protein substrate availability to the proteasome.

Eyestalk Ablation Has Little Effect on Actin and Myosin Heavy Chain Gene Expression in Adult Lobster Skeletal Muscles

The organization of skeletal muscles in decapod crustaceans is significantly altered during molting and development. Prior to molting, the claw muscles atrophy dramatically, facilitating their removal from the base of the claw. During development, lobster claw muscles exhibit fiber switching over several molt cycles. Such processes may be influenced by the secretion of steroid molting hormones, known collectively as ecdysteroids. To assay the effects of these hormones, we used eyestalk ablation to trigger an elevation of circulating ecdysteroids and then quantified myofibrillar mRNA levels with real-time PCR and myofibrillar protein levels by SDS-PAGE. Levels of myosin heavy chain (MHC) and actin proteins and the mRNA encoding them were largely unaffected by eyestalk ablation, but in muscles from intact animals, myofibrillar gene expression was modestly elevated in premolt and postmolt animals. In contrast, polyubiquitin mRNA was significantly elevated (about 2-fold) in claw muscles from eyestalk-ablated animals with elevated circulating ecdysteroids. Moreover, patterns of MHC and actin gene expression are significantly different among slow and fast claw muscles. Consistent with these patterns, the three muscle types differed in the relative amounts of myosin heavy chain and actin proteins. All three muscles also co-expressed fast and slow myosin isoforms, even in fibers that are generally regarded as exclusively fast or slow. These results are consistent with other recent data demonstrating co-expression of myosin isoforms in lobster muscles.

Molt cycle-dependent molecular chaperone and polyubiquitin gene expression in lobster.

Lobster claw muscle undergoes atrophy in correlation with increasing ecdysteroid (steroid molting hormone) titers during premolt. In vivo molecular chaperone (constitutive heat shock protein 70 [Hsc70], heat shock protein 70 [Hsp70], and Hsp90) and polyubiquitin messenger ribonucleic acid (mRNA) levels were examined in claw and abdominal muscles from individual premolt or intermolt lobsters. Polyubiquitin gene expression was assayed as a marker for muscle atrophy. Both Hsc70 and Hsp90 mRNA levels were significantly induced in premolt relative to intermolt lobster claw muscle, whereas Hsp70 mRNA levels were not. Hsp90 gene expression was significantly higher in premolt claw muscle when compared with abdominal muscle. Polyubiquitin mRNA levels were elevated in premolt when compared with intermolt claw muscle and significantly elevated relative to premolt abdominal muscle.

Osmotic induction of stress-responsive gene expression in the lobster Homarus americanus.

The American lobster, Homarus americanus, encounters osmotic stress throughout its life cycle. To understand the molecular basis of osmotic stress responses in vivo, we used homologous cDNA probes to characterize the mRNA patterns of lobster HSP70 (=70-kDa heat-shock protein), HSP90 (=90-kDa heat-shock protein), and polyubiquitin during hypo- and hyper-osmotic stress in abdominal muscle and hepatopancreas (a digestive tissue) at 30, 60, and 120 min of osmotic stress. Hypo- and hyper-osmotic stress significantly increased the levels of the mRNAs encoding HSP70 and HSP90 in abdominal muscle. Hyper-osmotic stress increased HSP90 mRNA levels in hepatopancreas, but hypo-osmotic stress did not. Both abdominal muscle and hepatopancreas exhibited significant changes in polyubiquitin gene expression during osmotic stress. In abdominal muscle, polyubiquitin mRNA levels increased during both hypo- and hyper-osmotic stress. Hepatopancreas, however, showed a significant elevation in polyubiquitin mRNA only during hypo-osmotic stress.

Ubiquitin-proteasome-dependent proteolysis in rainbow trout (Oncorhynchus mykiss): effect of food deprivation.

The ubiquitin-proteasome proteolytic pathway is a major route of protein degradation and of particular importance in muscle proteolysis in mammals. In this study, the beta proteasome subunit N3 and polyubiquitin genes of the rainbow trout, Oncorhynchus mykiss, were sequenced and tissue distribution of gene expression was examined. The effects of 14-day food withdrawal were assessed on the N3 subunit and polyubiquitin gene expression in terms of mRNA, 20S proteasome proteolytic activity and ubiquitin protein abundance in trout liver and muscle. Both sequences are highly conserved, and the rainbow trout ubiquitin amino acid sequence is identical to the mammalian protein. The proteasome beta subunit N3 has 92% similarity to the Xenopus sequence. Starvation halved the polyubiquitin mRNA level in liver but had no effect on muscle levels. No significant effect of food withdrawal was observed on the proteasome mRNA in liver or muscle. Food withdrawal decreased the 20S proteasome proteolytic activity and the abundance of ubiquitin protein in both muscle and liver. Co-regulation of the proteasome and ubiquitin was indicated by the high correlation ( R=0.924) between 20S activity and ubiquitin abundance. Overall, this study demonstrates that starvation down-regulates the ubiquitin-proteasome pathway, possibly highlighting differences in the regulation of protein turnover in poikilothermic and endothermic animals.

Thermal acclimation and stress in the American lobster, Homarus americanus: equivalent temperature shifts elicit unique gene expression patterns for molecular chaperones and polyubiquitin

Using homologous molecular probes, we examined the influence of equivalent temperature shifts on the in vivo expression of genes coding for a constitutive heat shock protein (Hsc70), heat shock proteins (Hsps) (Hsp70 and Hsp90), and polyubiquitin, after acclimation in the American lobster, Homarus americanus. We acclimated sibling, intermolt, juvenile male lobsters to thermal regimes experienced during overwintering conditions (0.4 +/- 0.3 degrees C), and to ambient Pacific Ocean temperatures (13.6 +/- 1.2 degrees C), for 4-5 weeks. Both groups were subjected to an acute thermal stress of 13.0 degrees C, a temperature shift previously found to elicit a robust heat shock response in ambient-acclimated lobsters. Animals were examined after several durations of acute heat shock (0.25-2 hours) and after several recovery periods (2-48 hours) at the previous acclimation temperature, following a 2-hour heat shock. Significant inductions in Hsp70, Hsp90, and polyubiquitin messenger RNA (mRNA) levels were found for the ambient-acclimated group. Alternatively, for the cold-acclimated group, an acute thermal stress over an equivalent interval resulted in no induction in mRNA levels for any of the genes examined. For the ambient-acclimated group, measurements of polyubiquitin mRNA levels showed that hepatopancreas, a digestive tissue, incurred greater irreversible protein damage relative to the abdominal muscle, a tissue possessing superior stability over the thermal intervals tested.

Contribution of the ubiquitin-proteasome pathway to overall muscle proteolysis in hypercatabolic patients

The influence of the gene expression of critical components of the cytoplasmic and lysosomal proteolytic pathways on the rate of protein degradation was evaluated in the leg skeletal muscle of 8 severely traumatized patients. Muscle proteolysis was determined as the intramuscular phenylalanine rate of appearance by L-[ring-2H5]phenylalanine infusion and the leg arteriovenous catheterization technique combined with muscle biopsy. Muscle mRNA levels of UbB polyubiquitin and cathepsin B were determined by reverse transcriptase-competitive polymerase chain reaction and expressed as a percent of the mRNA level of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the patients, individual values for UbB polyubiquitin mRNA levels directly correlated with the rate of muscle proteolysis (r = .76, P < .05), whereas no correlation (r = .10) was found between cathepsin B mRNA levels and proteolysis. Thus, after trauma, the rate of muscle proteolysis appears to be largely regulated by the ubiquitin-proteasome system at the level of gene transcription.

Isolation and characterization of a cDNA clone encoding polyubiquitin from the root nodule ofElaeagnus umbellata

A cDNA clone encoding polyubiquitin was isolated from a root nodule cDNA library of Elaeagnus umbellata Thunb. by differential hybridization, and its molecular aspects were characterized. The polyubiquitin clone pEuNOD-PUB1 has an insert size of 1642 bp and has the capacity to code for a 458 amino acid residue polyubiquitin protein. The derived amino acid sequence indicates that pEuNOD-PUB1 encodes a polyprotein consisting of six repeats of ubiquitin monomer, except for the last repeat, which has two additional amino acids (Asp-Phe) to be removed in the course of polyubiquitin processing into monomer. The molecular mass of ubiquitin monomer, consisting of 76 amino acids, was predicted to be 8524 Da, and the pI value was predicted to be 7.57. The nucleotide sequence of ubiquitin monomers from pEuNOD-PUB1 showed 73.1-86.0% sequence similarity with those from other organisms. The polyubiquitin mRNA content was four to six times higher in the root nodules than in the leaves and roots. In situ hybridization results showed polyubiquitin transcripts were strongly detected in the meristem zone, in infected cells of the fixation zone, and in the central vascular system. Genomic Southern hybridization revealed that polyubiquitin genes are present as a small multigene family in the genome of E. umbellata.Key words: Elaeagnus umbellata, root nodule, cDNA, polyubiquitin, Northern hybridization, in situ hybridization.

Differential Expression of Sugarcane Polyubiquitin Genes and Isolation of Promoters from two Highly-Expressed Members of the Gene Family

Summary To identify potential strong constitutive promoters, accumulation of polyubiquitin mRNA in sugarcane ( Saccharum hybrid) was studied using gene-specific probes from five sugarcane polyubiquitin cDNA clones. Based on comparisons of 3′ untranslated regions, these cDNAs can be grouped into four sub-groups within the sugarcane polyubiquitin gene family. RNA gel blot experiments indicated that these sub-groups differ substantially in constitutive expression levels and response to heat shock. Reverse northern experiments confirmed the relative magnitudes of constitutive expression in young leaves. One sub-group, homologous to cDNAs scubi241 and scubi511, was found to have the highest level of constitutive expression and to be little affected by heat shock. Genomic clones belonging to this sub-group were isolated; two of these clones, ubi4 and ubi9, were found to differ in the number of protein coding repeats but were highly similar in overall structure and in the sequence of DNA flanking the transcribed region. Both genes contain a single large intron located immediately upstream of the translation start codon. Transient expression experiments showed that the cloned promoters are sufficient to drive high levels of GUS expression in sugarcane suspension cells and in tobacco leaves.

Proteolytic Processes Underlying Molt-Induced Claw Muscle Atrophy in Decapod Crustaceans

The claw muscles of large-clawed decapod crustaceans undergo a programmed atrophy in preparation for molting, or ecdysis. This is mediated by five cytosolic proteinases organized into two proteolytic pathways: calcium-dependent and ubiquitin/proteasome-dependent. The calcium-dependent system consists of four calcium-dependent cysteine proteinases (CDPs I, IIa, IIb, and III; native masses 310, 125, 195, and 59 kDa, respectively) that completely degrade myofibrillar proteins and are activated in atrophic muscles. Immunological analysis shows that the active-site sequence in CDP IIa (60-kDa subunit mass) is similar to that in mammalian CDPs (calpains), and that CDP IIb is homologous to a calpain-like gene isolated from Drosophila cDNA libraries. Increased intracellullar Ca2+ stimulates proteolysis in situ , indicating CDPs play an important role in muscle protein catabolism. The ubiquitin/proteasome-dependent system involves the ATP-dependent conjugation of multi-ubiquitin chains to protein by ubiquitin-conjugating enzymes. This acts as a signal for substrate degradation by the 26S proteasome, a multi-subunit complex consisting of a 20S proteasome catalytic “core” and two PA700 (19S) regulatory complexes. Polyubiquitin mRNA, ubiquitin-protein conjugates, and 20S proteasome are elevated about 5-, 8-, and 2-fold, respectively, during atrophy. A heat-induced form of the 20S proteasome hydrolyzes myosin, troponin, and tropomyosin to large fragments in vitro . Biochemical studies identified the branched-chain amino acid-preferring (BrAAP) activity, one of six distinct catalytic components in the complex, as the activity that carries out these initial cleavages. These results indicate that the ubiquitin/proteasome pathway is involved, but its precise role remains to be resolved.

Sequence analysis and ozone-induced accumulation of polyubiquitin mRNA in Pinus sylvestris

On sait que les plantes reagissent a l'ozone a l'echelle moleculaire par l'expression de genes relies a un mecanisme de defense. Les auteurs ont crible de facon differentielle une banque d'ADNc de pin sylvestre (Pinus sylvestris L.) en utilisant l'ARN poly(A) + marque et transcrit a l'inverse. Cet ARN fut isole de semis de pin sylvestre traites a l'ozone et de semis non traites. De plus, le clone d'ADN decoulant du plus long traitement d'induction a l'ozone (O 3 I-2,3) fut caracterise par sequencage de l'ADN et par hybridation Northern. Le sequencage d'ADN a permis de determiner que ce clone etait en fait un ADNc de polyubiquitine forme de 10 repetitions de 228 acides nucleiques chacune. Ce clone est le plus long ADNc de polyubiquitine caracterise chez les plantes et le premier clone d'ADNc representant l'ensemble de la zone transcrite a etre rapporte pour un gymnosperme. Les analyses d'hybridation Northern ont revele une augmentation transitoire pour deux especes de polyubiquitine de 2,5 et 1,6 kb, tandis que la quantite pour l'espece de 1,1 kb restait constante apres le traitement a l'ozone des semis de pin sylvestre. Ces donnees indiquent que le systeme des ubiquitines pourrait etre implique au niveau de la conjuguaison des proteines qui sont endommagees par l'ozone.

Independent modulation of Arabidopsis thaliana polyubiquitin mRNAs in different organs and in response to environmental changes

The highly conserved protein ubiquitin is encoded by five polyubiquitin genes in Arabidopsis thaliana ecotype Columbia that have been divided into two subtypes, the UBQ3/UBQ4 subtype and the UBQ10/UBQ11/UBQ14 subtype. Northern analysis using gene-specific oligonucleotides as hybridization probes and enzyme activity measurements from transgenic plants expressing beta-glucuronidase (GUS) under the control of individual polyubiquitin 5' flanking regions were used to determine the development and environmental regulation of polyubiquitin transcription and mRNA accumulation. Polyubiquitin mRNA levels within and between subtypes were independently modulated. UBQ3 mRNA levels were three-fold higher than UBQ4 mRNA levels in vegetative organs, but only two-thirds of the UBQ4 mRNA levels in flowers. UBQ3 mRNA was modulated by dark/light treatments, while mRNAs from UBQ and all members of the other subtype were unaffected. Similarly, within the UBQ10/UBQ11/UBQ14 subtype, UBQ11/UBQ14 mRNAs were modulated differently in seedlings after a two-hour heat-shock treatment. Among all the polyubiquitin genes, UBQ10 mRNA level was the most constant in all organs and environmental conditions examined. Transgenic plants transformed with a UBQ10 5' flanking region::GUS gene contained higher levels of GUS activity than transgenic plants expressing GUS under the control of UBQ3 5' flanking regions. In conclusion, the relative abundance of different Arabidopsis polyubiquitin mRNAs, even those produced from highly similar genes within a subtype, appears to be modulated independently in response to developmental and environmental cues.

Gene expressions of ubiquitin and hsp70 following focal ischaemia in rat brain.

Expression of genes coding for ubiquitin and heatshock protein (hsp) 70 were examined by in situ hybridization using a rat model with permanent occlusion of the distal middle cerebral artery (MCA). Only polyubiquitin (UbC) mRNA increased markedly following ischaemia in the central zone of the MCA territory of the neocortex. UbC gene expression reached the maximum level 4 h post-occlusion and remained elevated at 24 h. UbC expression was retarded slightly compared with that of the hsp70 gene. UbB and Ub-S30 were expressed at almost similar levels in both the ischaemic and non-ischaemic hemispheres. These results indicated that UbC probably has the most stress-inducible characteristics among the three ubiquitin genes.

Sequence analysis and ozone-induced accumulation of polyubiquitin mRNA in &lt;I&gt;Pinus sylvestris&lt;/I&gt;

Sequence analysis and ozone-induced accumulation of polyubiquitin mRNA in &lt;I&gt;Pinus sylvestris&lt;/I&gt;

Polyubiquitin in crustacean striated muscle: increased expression and conjugation during molt-induced claw muscle atrophy

The claw muscles of decapod crustaceans undergo a molt-induced atrophy to facilitate withdrawal of the claws at ecdysis. Polyubiquitin expression, as well as the levels of ubiquitin conjugates, a ubiquitin-conjugating enzyme involved in the ATP/ubiquitin-dependent proteolytic pathway (crustacean E2 16 kDa homolog of Drosophila UbcDl), and proteasome, were examined to determine the role of ATP/ubiquitin-dependent proteolysis in the enhanced degradation of myofibrillar proteins during muscle atrophy. A partial-length clone (1.7 kb) of polyubiquitin was isolated from a lobster muscle cDNA library; the 5′ end lacked the 5′ untranslated region (UTR) and the beginning of the first ubiquitin monomer, while the 3′ end contained the terminal ubiquitin monomer and 3′ UTR. The deduced amino acid sequence was 100% identical with that from Manduca, Drosophila, and human. In land crab claw muscle, the polyubiquitin mRNA (2.7 kb) increased about 5-fold and ubiquitin-protein conjugates (> 200 kDa) increased about 8-fold during atrophy. In contrast, the level of a ubiquitin-conjugating enzyme (E2 16 kDa) remained unchanged. The proteasome, which constitutes the catalytic core of the ATP/ubiquitin-dependent proteinase complex, increased about 2-fold during proecdysis, reaching its highest level immediately before ecdysis. These results suggest that the ATP/ubiquitin-dependent proteolytic pathway contributes to the changes in protein metabolism that occur during molt-induced muscle atrophy.

Expression of ubiquitin genes in Chlamydomonas reinhardtii: involvement in stress response and cell cycle

Applying different stresses (heat shock, photoinhibition, and chilling) to the unicellular green alga Chlamydomonas reinhardtii led to a characteristic transcription pattern of mRNAs encoding ubiquitin extension proteins [0.7-kb transcript(s)] as well as polyubiquitin (2.3-kb transcript). Heat shock resulted in an increase in the amount of polyubiquitin mRNA (up to tenfold compared with control cells). Chilling in the light led to a sevenfold increase in the amount of 2.3-kb transcript and to a twofold increase in the amount of 0.7-kb mRNA(s), whereas a less pronounced effect on the level of the polyubiquitin transcript was observed after applying either chilling in darkness or photoinhibition. The latter stress, however, led to a dramatic decrease in mRNAs encoding ubiquitin extension proteins (down to 23% of control cells). Experiments performed with a temperature-sensitive cell-cycle mutant of C. reinhardtii showed that the 2.3-kb polyubiquitin mRNA was no longer transcribed when cells were shifted to a non-permissive temperature, and thus were blocked in their vegetative cell cycle. This leads to the assumption that the expression of the corresponding gene is necessary for completion of the C. reinhardtii vegetative cell cycle.

Coordinated Induction of the Ubiquitin Conjugation Pathway Accompanies the Developmentally Programmed Death of Insect Skeletal Muscle

The developmentally programmed cell death of abdominal intersegmental muscles in the tobacco hawk-moth Manduca sexta is coincident with a 10-fold induction of the polyubiquitin gene as a hormonally regulated event (Schwartz, L. M., Myer, A., Kosz, L., Engelstein, M., and Maier, C. (1990) Neuron 5, 411-419). Solid phase immunochemical assays measuring intersegmental muscle pools of free and conjugated ubiquitin reveal that the induction of polyubiquitin mRNA is accompanied by a proportional increase in total ubiquitin polypeptide. Ubiquitin conjugate pools increase 10-fold at eclosion, during which loss of muscle protein mass is maximum. A smaller but measurable increase in ubiquitin conjugates is observed earlier in pupal development coincident with a modest enhanced degradation of myofibrillar proteins. Accumulation of ubiquitin conjugates is accompanied by induction in the pathway for polypeptide ligation, including the activating enzyme (E1), several carrier protein (E2) isoforms, and ubiquitin:protein isopeptide ligase (E3). Both accumulation of ubiquitin polypeptide and the enzymes of the conjugation pathway are subject to regulation by declining titers of the insect molting hormone 20-hydroxyecdysone, which signals onset of programmed cell death in the intersegmental muscles. Thus, programmed cell death within the intersegmental muscles is accomplished in part by stimulation of the ubiquitin-mediated degradative pathway through a coordinated induction of ubiquitin and the enzymes responsible for its conjugation to yield proteolytic intermediates. This suggests enzymes required for ubiquitin conjugation may represent additional genes recruited for developmentally programmed death.

Protein ubiquitination in the posterior silk glands of Bombyx mori

Ubiquitin gene expression and the ubiquitination of proteins in the posterior silk glands (PSG) of B. mori were analyzed developmentally with respect to fibroin synthesis and degeneration. Two ubiquitin transripts are expressed throughout larval stages, and the level of each transcript is regulated differently. The larger transcript, a polyubiquitin mRNA, was abundant during the molt stage, while levels of the smaller ubiquitin transcript increased immediate after molting. Only a single 65kDa ubiquitinated protein was detected late in the 5th instar, and the amount increased up to spinning stage. This ubiquitinated protein may participate in the PSG degeneration.