Dorothy M. Skinner

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.

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

1. The morphological changes undergone during the molt cycle by the integumentary tissue of the land crab, Gecarcinus lateralis, have been described.2. The time course of limb regeneration and gastrolith formation has been correlated with the morphological changes of the integumentary tissue. The period of premolt activity during which limb regeneration and gastrolith formation occurs precedes the changes in the integumentary tissues and has, therefore, been called DO.3. The oxygen consumption of the integumentary tissues has been measured at each stage of the molt cycle. It has been found to increase at the time of synthesis of the exocuticle. The effects of cyanide, dinitrophenol and added substrates on the oxygen consumption of the integumentary tissue have been studied.

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

1. Loss of 6 to 8 pereiopods or chelipeds triggers precocious molts in a number of marine crabs including the green crab, Carcinus maenas, the blue crab, Callinectes sapidus, and the fiddler crabs, Uca pugnax and U. pugilator, but not in the spider crab, Libinia emarginata. Mortality rates are negligible compared to those of animals induced to molt by eyestalk removal.2. Precocious molts call be elicited in the land crab, Gecarcinus lateralis, by the loss of 5 to 8 appendages (pereiopods and chelipeds) but the loss of all 10 appendages inhibits molting. Loss of a cheliped which in Gecarcinus may have a mass ten times greater than a pereiopod is no more effective than loss of a walking leg.3. The size of the regenerates formed in Gecarcinus is reduced by one-third from normal size when front 1 to 6 pereiopods are lost, and by one-half when 8 limbs are regenerated.4. When one or more partially regenerated limbs is removed before a certain critical time in the premolt period (Stage D0?) the animal re-regener...

Molting in land crabs: stimulation by leg removal.

If the Bermuda land crab, Gecarcinus lateralis, loses numerous walking legs or both chelipeds, it undergoes almost immediate preparations for molting with attendant limb regeneration. Injections of the arthropod-molting hormone, ecdysterone, have no effect in either intact animals or those missing legs.

Molt cycle-associated changes in calcium-dependent proteinase activity that degrades actin and myosin in crustacean muscle

Abstract The role of calcium-dependent proteinase (CDP) in the proecdysial atrophy of crustacean claw muscle has been investigated. During atrophy the molar ratio of actin to myosin heavy chain decreased 31%, confirming earlier ultrastructural observations that the ratio of thin:thick myofilaments declined from 9:1 to 6:1 (D. L. Mykles and D. M. Skinner, 1981, J. Ultrastruct. Res. , 75, 314–325) . The release of TCA-soluble material in muscle homogenates at neutral pH was stimulated by Ca 2+ and completely inhibited by EGTA. The specific degradation of the major myofibrillar proteins (actin, myosin heavy and light chains, paramyosin, tropomyosin, troponin-T, and troponin-I) was demonstrated by SDS-polyacrylamide gel electrophoresis. Proteolytic activity was more than twofold greater in proecdysial muscle homogenates. Degradation of myofibrillar proteins was inhibited by EGTA, and the two inhibitors of cysteine proteinases, leupeptin and antipain, but not pepstatin, an inhibitor of aspartic proteinases. Unlike CDPs from vertebrate muscle, the CDP(s) in crab claw muscle degrades actin and myosin in addition to other myofibrillar proteins.

Preferential loss of thin filaments during molt-induced atrophy in crab claw muscle

The closer muscle of the claw of a land crab Gecarcinus lateralis consists of slow, or tonic, fibers that undergo a sequential atrophy and restoration during each molting cycle. We have examined the ultrastructural organization of claw fibers during the process of protein degradation that takes place in proecdysial muscle atrophy. The myofibril cross-sectional area is reduced fourfold by dissolution of peripheral myofilaments and by focal erosion within the fibrils. As degradation continues, there is a preferential loss of thin filaments relative to thick filaments: (1) the average distance between thick filaments decreases from 59 to 45 nm, causing a 72% increase in packing density of thick filaments in cross section and (2) the relative numbers of thin and thick filaments decrease from an average ratio of 9:1 to 6:1. Despite these significant structural changes, the general organization of the sarcomere is unaltered; the membrane surface density of the sarcoplasmic reticulum and the A-band length remain unchanged. Although there is autophagy of some mitochondria, others retain their normal appearance, as do the nuclei. These data suggest that the specific degradation of contractile proteins in procedysial atrophic muscle includes the differential breakdown of thin versus thick filaments.

INTERACTIONS BETWEEN MOLTING AND REGENERATION IN THE LAND CRAB

1. Following the loss of one or more primary regenerates from the land crab, Gecarcinus lateralis, before a critical stage of the molt cycle (probably D1, early), secondary regenerates form to replace those lost.2. Although loss of at least five limbs from an intermolt animal is required to initiate molting preparations (Skinner and Graham, 1972) loss of only one primary regenerate, prior to the critical time, is sufficient to inhibit these preparations.3. Following the loss of one or more primary regenerates before a critical time, the rate of growth and the synthesis of DNA in other primary regenerates that remain in situ decrease or cease temporarily (10 to 14 days).4. The precise stage of the animal in the molt cycle determines whether growth and/or DNA synthesis decrease or cease in the remaining primary regenerates. Loss of regenerates early in D0 (R = 7-10) causes a decrease; loss of regenerates later in D0 or early D1 (R = 10-17) causes complete inhibition.5. The duration of the premolt period is ...

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

Caiinectes sapidus is the only true crab (brachyuran) whose pattern of ecdyste roid titers has been described as departing from the pattern seen in other decapods. While ecdysteroids in other crabs reach a peak just prior to ecdysis, those of C. sapidus were claimed to reach their maxima after ecdysis. The data reported here challenge these findings. We have measured ecdysteroids in hemolymph, ovaries, and whole animal extracts of blue crabs using a radioimmunoassay. In hemolymph and whole animals, ecdysteroid levels rose during premolt to a maximum at stage D3 . Ecdysteroids declined rapidly from late premolt stage D4 through postmolt stage A2, increased slightly at postmolt stage B, and returned to low levels where they remained during intermolt stage C. Ecdysteroid levels in males and immature fe males were not significantly different but mature females, having reached a terminal anecdysis, had significantly lower ecdysteroid levels. Ovaries of mature females ac cumulated ecdysteroids during vitellogenesis while the concentration of ecdysteroids in hemolymph was low.

Triplex DNA in plasmids and chromosomes

Circular plasmids containing pyrimidine purine tracts can form both inter-and intramolecular triplexes. Addition of poly(dTC) to plasmid pTC45, which contains a (TC)45.(GA)45 insert, results in intermolecular triplex formation. Agarose-gel electrophoresis gives rise to many well-resolved bands, which correspond to 1, 2, 3, 4... plasmid molecules attached to the added pyrimidine strand. In the electron microscope these complexes appear as a rosette of petals. The mobility of these triplex-containing complexes can be retarded by the addition of a triplex-specific monoclonal antibody, Jel318. Intramolecular triplex formation can be demonstrated at pH 5 in pTC45 and also in pT463-I, a plasmid containing a segment of a crab satellite DNA with both (G)n.(C)n and (TCC)n.(GGA)n inserts. However, although the intermolecular triplex remains stable for some time at pH 8, intramolecular triplex formation only occurs at low pH. Triplexes can also be detected by an immunoblotting procedure with Jel318. This unfamiliar structure is readily demonstrated in eukaryotic extracts, but not in cell extracts from Escherichia coli. Triplexes may thus be an inherent feature of eukaryotic chromosome structure.

Atrophy of crustacean somatic muscle and the proteinases that do the job. A review

As much as 60% of the muscle in the propodus of both chelae of some brachyurans atrophies during proecdysis and is restored during metecdysis. We describe here the structural changes that occur during atrophy of such chelae muscles as well as atrophy of somatic muscle from a number of species of Crustacea in response to one or more of several physiological stimuli. Atrophy is not random and whole fibers are not lost; the remaining cellular structures provide a framework into which newly synthesized myofibrillar proteins are packed during metecdysis. As seen in electron micrographs as well as in gel electropherograms, more thin filament proteins (actin, troponin, and tropomyosin) are degraded when compared to those from thick filaments