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D M Skinner

Publications and source records attributed to D M Skinner.

At least 37 records · Page 2Linked to original sources
PubMed1983

Ca2+-dependent proteolytic activity in crab claw muscle. Effects of inhibitors and specificity for myofibrillar proteins.

The claw closer muscle of the Bermuda land crab, Gecarcinus lateralis, undergoes a sequential atrophy and restoration during each molting cycle. We describe here the role of Ca2+-dependent proteinases in the turnover of myofibrillar protein in normal anecdysial (intermolt) claw muscle. Crab Ca2+-dependent proteinase degrades the myofibrillar proteins actin, myosin heavy and light chains, paramyosin, tropomyosin, and troponin-T and -I. Ca2+-dependent proteinase activity in whole homogenates and 90,000 X g supernatant fractions from muscle homogenates has been characterized with respect to Ca2+ requirement, substrate specificity, and effects of proteinase inhibitors. The enzyme is inhibited by antipain, leupeptin, E-64, and iodoacetamide; it is insensitive to pepstatin A. The Ca2+-dependent proteinase is a sarcoplasmic cysteine proteinase that shows maximal activation at 1 mM Ca2+ at neutral pH. Since approximately 28% of the activity remains at 1.5 microM Ca2+, the enzyme is partially active at physiological Ca2+ concentrations. The specificity of crab Ca2+-dependent proteinase was examined with native myosin with normal ATPase activity as well as with radioiodinated myosin and radioiodinated hemolymph proteins. Hydrolysis of 125I-myosin occurs in two phases, both Ca2+-dependent: 1) heavy chain (Mr = 200,000) is cleaved into four large fragments (Mr = 160,000, 110,000, 73,000, 60,000) and numerous smaller fragments; light chain (Mr = 18,000) is cleaved to a 15,000-Da fragment; 2) the fragments produced in the first phase are hydrolyzed to acid-soluble material. Although radioiodinated native hemolymph proteins are not susceptible to the Ca2+-dependent proteinase, those denatured by carboxymethylation are degraded. These data suggest that crab Ca2+-dependent proteinase is involved in turnover of myofibrillar protein in normal muscle and muscle undergoing proecdysial atrophy.

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PubMed1983

An inverted repeat borders a fivefold amplification in satellite DNA.

One variant of a complex satellite DNA of the Bermuda land crab is significantly longer than the average repeat unit of the satellite. The extra DNA in the variant is accounted for by a fivefold tandem amplification of a 0.142-kilobase sequence. The amplified sequence is bounded by a tetranucleotide inverted repeat; the upstream arm of the inverted repeat is missing from two other variants of the satellite. The latter variants contain only one copy of a sequence that is closely related to the amplified sequence. By contrast, in several satellite DNA's of other organisms, extra DNA is inserted.

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PubMed1981

Irreversible denaturation mapping of a pyrimidine-rich domain of a complex satellite DNA.

The highly complex G + C-rich satellite DNA of the Bermuda land crab Gecarcinus lateralis has been studied by denaturation mapping. Following digestion of the satellite with EndoR.Eco RI, the major 2.07-kilobase pair (kbp) basic repeating unit and a minor 4.14-kbp fragment were exposed to 254 nm light in the presence of silver ions, conditions which resulted in essentially irreversible denaturation of regions rich in adjacent pyrimidines by the formation of pyrimidine dimers. The positions and sizes of the denatured regions were determined in electron micrographs of partially denatured 2.07-kbp and 4.14-kbp fragments spread in the presence of formamide. After 15 min exposure to UV, 90% of the 2.07-kbp fragments had a denaturation bubble averaging 0.17 kbp centered around one-third (0.64 kbp) the total length; 20% exhibited another in the region from 1.8 kbp to 2.07 kbp. Similarly, about 90% of the 4.14-kbp fragments had denatured regions centered at 0.64 kbp and 2.75 kbp and 20% of the fragments had denaturation bubbles in regions centered at 1.92 kbp and 3.9 kbp. The positions of the denaturation bubbles in the 4.14-kbp fragments support restriction enzyme mapping evidence that it is a dimer of the 2.07-kbp fragment arranged head to tail. Sequencing data show that the predominant sequence of a 0.29-kbp region centered around 0.64 kbp in the basic repeat unit is 49% A + T and that 42% of the bases are adjacent TTs and CTs capable of dimerization under the conditions used.

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PubMed1980

Evidence for nonrandom alterations in a fraction of the highly repetitive DNA of a eukaryote.

Although the DNA of the red crab Geryon quinquedens has no patent satellites, a large fraction (approximately 40%) is highly repetitive. Treatment of total DNA by Hind III produces fragments comprising 5-6% of the genome. While the sizes of some of these fragments form an arithmetic series based on an 81 bp repeating unit, the amounts of the multimers differ significantly from distributions observed for multimeric series in the DNAs of other eukaryotes. In red crab DNA, the amounts of some of the multimers suggest that they may have undergone as much as four times the divergence as the others. Other data, however, are more compatible with the conclusion that there has been selective amplification of segments of highly repeated DNA which results in the enhanced amount of specific multimers. These results indicate the presence of a nonrandom process in the evolution of the highly repetitive DNA. Selective mutation alone seems insufficient to explain these results.

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PubMed1980

Selective amplification of variants of a complex repeating unit in DNA of a crustacean.

The nucleotide sequence of the repeating unit of a fraction of the highly repetitive DNA of the red crab, Geryon quinquedens, is reported. Treatment of total DNA with HindIII nuclease produced an 81-base-pair monomer and multimers to the size of an octamer. Several of the multimers contained large amounts of fragments of variant sequences, which cannot easily be explained by random mutation alone. That the alterations were not random was corroborated by divergence measurements made on the distribution of Hha I nuclease sites within several multimers. The analyses showed that a fraction of each of them is characterized by 4% divergence, while the amounts of dimer, tetramer, and octamer suggest that they have undergone 2-4 times more divergence than that. These results, coupled with the data on sequence variants that are more prevalent in the dimer, indicate that amplification of divergent repeating units could easily explain enhanced amounts of selected multimers.

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PubMed1979

Interspersion of highly repetitive DNA with single copy DNA in the genome of the red crab, Geryon quinquedens.

Kinetic analysis of the reassociation of 420 nucleotide (NT) long fragments has shown that essentially all of the repetitive sequences of the DNA of the red crab Geryon quinquedens are highly repetitive. There are negligible amounts of low and intermediate repetitive DNAs. Though atypical of most eukaryotes, this pattern has been observed in all other brachyurans (true crabs) studied (1,2). The major repetitive component is subdivided into short runs of 300 NT and longer runs of greater than 1200 NT while the minor component has an average sequence length of 400 NT. Both components reassociate at rates commonly observed for satellite DNAs. Unique among eukaryotes the organization of the genome includes single copy DNA contiguous to short runs (approximately 300 NT) of both repetitive components. Although patent satellites are not present, subsets of the repetitive DNA have been isolated by either restriction endonuclease digestion or by centrifugation in Ag+ or Hg2+/Cs2SO4 density gradients.

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PubMed1978

The primary sequence of a crustacean satellite DNA containing a family of repeats.

The repeating sequence of isopycnic twin Satellite II of the hermit crab, Pagurus pollicaris, has been determined. More than 95% of the primary sequence of the light strand is composed of (CAG)nCTGCACT. The only detectable variation on the repeating sequence is the number of CAGs included in each repeat which varies from 3-12. The sequence of Satellite II differs markedly from that of its twin, whose light strand is greater than 95% CCTA (Skinner et al., 1974). The absence from Satellite II of repeats where (CAG)n less than or equal to 2 further defines the constraints on a recent model for the origin of satellites (Smith, 1976).

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PubMed1977

The interactions of the separated strands of satellite DNAs with other DNAs: 1. Conditions for associations of the alpha-satellite of the guinea pig with heterologous double-stranded DNAs.

The separated H- and L-strands of the alpha-satellite of the guinea pig, Cavea porcellus, recovered from centrifugation in alkaline CsC1 gradients, from complexes with 7 different double-stranded (ds) DNAs including those of 1 bacteriophage, 2 prokaryotes, 2 invertebrates and 2 mammals. The complexes are not artifacts due to in vitro labeling of the satellite, methods of collection, the presence of divalent cations, or the fact that trace amounts of single-stranded (ss) DNAs are used. More complex dsDNAs, such as that recovered from nicked RF M13, do not associate with dsDNAs.

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