THE STRUCTURE AND METABOLISM OF A CRUSTACEAN INTEGUMENTARY TISSUE DURING A MOLT CYCLE

Volume: 123Start Page: 635End Page: 64

Denaturants or Cosolvents Improve the Specificity of PCR Amplification of a G + C-Rich DNA Using Genetically Engineered DNA Polymerases

We describe conditions that improve the specificity of amplification of a G + C-rich (57% G + C) DNA by PCR. Under standard conditions a 368-bp segment of the approx. 2.1-kb repeat unit of a satellite DNA that accounts for approx. 3% of the genome of the Bermuda land crab, Gecarcinus lateralis, was not amplified specifically. To establish optimal conditions for amplification of the segment of the G + C-rich satellite, we used two genetically engineered enzymes, AmpliTaq DNA polymerase and AmpliTaq DNA polymerase. Stoffel fragment (SF), and a number of denaturants or co-solvents. In the absence of denaturants or co-solvents, amplified products of both enzymes contained non-specific bands upon gel electrophoresis. Addition of certain denaturants or co-solvents to PCR mixtures resulted in the production of the single specific band of the expected size. Reagents that improved specificity of the amplified product were formamide, glycerol, DMSO, Tween-20 and NP-40; on the other hand, urea, ethanol and 1-methyl-2-pyrrolidone (NMP) inhibited amplification. Of the two enzymes, SF was more specific and efficient. The products of AmpliTaq DNA polymerase included one or more extra bands, even in the presence of denaturants or co-solvents, except for glycerol or DMSO

ECDYSTEROID TITERS DURING THE MOLT CYCLE OF THE BLUE CRAB RESEMBLE THOSE OF OTHER CRUSTACEA

Volume: 165Start Page: 321End Page: 32

Cytoplasmic Localization of Transcripts of a Complex G+C-Rich Crab Satellite DNA

The primary sequence and higher order structures of a G+C-rich satellite DNA of the Bermuda land crab Gecarcinus lateralis have been described previously. The repeat unit of the satellite is approximately 2.1 kb. In exploring a possible function for this satellite, we asked whether it is transcribed. As a probe for transcripts, we used a segment of DNA amplified from a 368 bp EcoRI fragment from the very highly conserved 3′ end of the satellite DNA. During polymerase chain reaction (PCR) amplification, the probe was simultaneously either radiolabeled or biotinylated. Tissue- and stage-specific transcripts were observed when blots of poly(A)+ mRNAs recovered from polysomes isolated from crab tissues [including midgut gland (hepatopancreas), limb bud, and claw muscle] were probed with the satellite DNA fragment. The presence of satellite transcripts in polysomal mRNAs is strong evidence that the transcripts had reached the cytoplasm. To corroborate the presence of transcripts in the cytoplasm, we investigated in situ hybridization of satellite probes with RNAs in tissue sections. Biotinylated satellite DNA probes were applied to sections of midgut gland, limb bud papilla, ovary, or testis of anecdysial crabs. Retention of RNAs in tissue sections was improved by UV-irradiation prior to hybridization. Transcripts were abundant in the cytoplasm of all tissues except testis. Sections of crab midgut gland treated with RNase A prior to hybridization and sections of mouse pancreatic tumor served as controls; neither showed any signals with the probe

LOSS OF LIMBS AS A STIMULUS TO ECDYSIS IN BRACHYURA (TRUE CRABS)

Volume: 143Start Page: 222End Page: 23

Effects of 20‐hydroxyecdysone on Protein Synthesis in Tissues of the Land Crab Gecarcinus Lateralis

The endocrine regulation of molting was investigated in the crab Gecarcinus lateralis by treating tissues from anecdysial (intermolt) animals with the arthropod molting hormone 20‐hydroxyecdysone (20HE) in vitro and observing changes in the incorporation of [35S]‐methionine into proteins. Although total protein synthesis was not affected by levels of 20HE ranging from 10−9 to 10−5 M, synthesis of five out of 30 protein bands was significantly stimulated at hormone concentrations of 10−9−10−7 M, the latter characteristic of proecdysis. Increases were as large as eightfold. Synthesis was not significantly inhibited for any of the integumentary proteins. Treatment of integumentary tissues with 10−5 M 20HE (approx. 100 times greater than the highest endogenous concentration) had less effect than physiological concentrations. The dose response varied substantially among integumentary proteins. In some instances, the effects were similar to changes in synthesis that occur in vivo during specific stages of proecdysis (Stringfellow and Skinner, Dev. Biol., 128: 97–110, \u2788). Midgut gland (hepatopancreas) was also treated with 20HE in vitro. Treatment with 20HE significantly inhibited total protein synthesis in tissue from males, but not in that from females. This treatment inhibited the synthesis of a number of the 32 protein bands examined in both sexes. Synthesis of six protein bands was significantly inhibited in males, while synthesis of three different bands was significantly inhibited in females. This inhibitory action of 20HE is consistent with the atrophy of the midgut gland that occurs during proecdysis (Gibson and Barker, Oceanogr. Mar. Biol. Annu. Rev., 17: 285–346, 79)

Molt-Cycle Correlated Patterns of Synthesis of Integumentary Proteins in the Land Crab Gecarcinus Lateralis

We describe here an in vitro investigation of the formation and dissolution of the crustacean exoskeleton by the integumentary tissues of the Bermuda land crab Gecarcinus lateralis. We focus on the synthesis of proteins during the formation of a new exoskeleton and the degradation of an old one. Tissues of animals at different stages of the molt cycle were labeled in vitro with radioamino acids following which proteins were analyzed by a number of methods. From their patterns of synthesis, crab integumentary proteins (CIPs) may be classified into five groups. Group I CIPs include many over a wide range of molecular weights that are synthesized at a relatively constant rate throughout the molt cycle and are designated housekeeping proteins. The synthesis of a subset of such housekeeping CIPs (Group II) increases from two- to fivefold during proecdysis when formation of the epicuticle and exocuticle of the new exoskeleton is in progress. Group III CIPs are synthesized intermittently from very early proecdysis, when degradation of the old exoskeleton begins, and throughout the remainder of proecdysis. Groups IV and V first appear at stages in the molt cycle that suggest that they may be structural components of the new exoskeleton. Group IV CIPs, which are synthesized at the time of epi- and exocuticle formation, have a wide range of sizes. Group V CIPs, which comprise several very large proteins, are synthesized in stage B of metecdysis at the time when the formation of the endocuticle begins. A number of CIPs cross-react strongly with polyclonal antibodies to cuticle proteins of pupae and larvae of the tobacco hornworm Manduca sexta, indicating that exoskeletal/cuticle proteins of these two classes of arthropods, the Insecta and Crustacea, share epitopes. Whether the common epitopes relate to amino acid sequences or structural properties remains to be determined

Proteins of Crustacean Exoskeleton II: Immunological Evidence for Their Relatedness to Cuticular Proteins of Two Insects

The exoskeletons of crustaceans and cuticles of insects are characterized by an abundance of proteins with acidic pIs and Mrs 31 kDa or smaller. Immunochemical data described here further demonstrate the similarities of such proteins among representatives of these two major classes of Arthropoda. Proteins extracted from the four individual layers of the exoskeletons of Bermuda land crabs Gecarcinus lateralis at different stages of the intermolt cycle and from exuviae, the partially degraded old exoskeleton that is cast at ecdysis, were analyzed on Western blots with polyspecific polyclonal antibodies against either larval cuticular proteins (anti‐LCP) or pupal cuticular proteins (anti‐PCP) of the tobacco hornworm Manduca sexta, or against two pairs of larval cuticular proteins from third instar larvae (anti‐L3CPs) of Drosophila melanogaster. M. sexta anti‐LCP cross‐reacted with many of the crab proteins extracted at all stages of the intermolt cycle. It reacted with more high Mr proteins from late proecdysial exoskeleton and exuviae than with such proteins from anecdysial or early proecdysial exoskeletons, probably because the larger Mr proteins are enriched when the lower Mr proteins are degraded during proecdysis. M. sexta anti‐PCP cross‐reacted hardly at all with the crab proteins. Both D. melanogaster anti‐L3CPs cross‐reacted with crab epicuticular proteins at all stages of the intermolt cycle and from exuviae. Anti‐L3CP 3 + 4 reacted even more intensely and with many more proteins from the three inner layers of the exoskeleton than did anti‐L3CP 1+2. Controls included 2D gels with rabbit anti‐ovalbumin to detect glycoproteins containing mannose and N‐acetyl‐glucosamine; it cross‐reacted with only one protein in membranous layer of an anecdysial crab, while rabbit anti‐fluorescein reacted with none. By contrast, the intensity and specificity of the cross‐reactions of crab exoskeletal proteins with antibodies against cuticular proteins of members of two orders of insects suggest that at least some cuticular proteins in the two arthropod Classes, Crustacea and Insecta, are evolutionarily conserved. Published 1993 Wiley‐Liss, Inc