T. Meedel - Academia.edu (original) (raw)

Papers by T. Meedel

Research paper thumbnail of Development of the Ascidian Embryo: Cell Fate Specification by Autonomous and Inductive Processes

... (D) The b7. B blastomeres also give rise to four undefined cells in the posterior, dorsal reg... more ... (D) The b7. B blastomeres also give rise to four undefined cells in the posterior, dorsal region of the tail (37). Page 290. ... Van Bene, den and Julin were the first to investigate the relationship between cells of the ascidian embryo and their later developmental fates (101). ...

Research paper thumbnail of Ascidian Larva Reveals Ancient Origin of Vertebrate-Skeletal-Muscle Troponin I Characteristics in Chordate Locomotory Muscle

Molecular Biology and Evolution, 2003

Ascidians are protochordates related to vertebrate ancestors. The ascidian larval tail, with its ... more Ascidians are protochordates related to vertebrate ancestors. The ascidian larval tail, with its notochord, dorsal nerve cord, and flanking rows of sarcomeric muscle cells, exhibits the basic chordate body plan. Molecular characterization of ascidian larval tail muscle may provide insight into molecular aspects of vertebrate skeletal muscle evolution. We report studies of the Ci-TnI gene of the ascidian Ciona intestinalis, which encodes the muscle contractile regulatory protein troponin I (TnI). Previous studies of a distantly related ascidian, Halocynthia roretzi, showed that different TnI genes were expressed in larval and adult muscles, the larval TnI isoforms having an unusual C-terminal truncation not seen in any vertebrate TnI. Here we show that, in contrast with Halocynthia, Ciona does not have a specialized larval TnI; the same TnI gene that is expressed in the heart and body-wall muscle of the sessile adult is also expressed in embryonic/ larval tail muscle cells. Moreover the TnI isoform produced in embryonic/larval muscle is identical to that produced in adult body-wall muscle, i.e., a 182-residue protein with the characteristic chain length and overall structure of vertebrate skeletal muscle TnI isoforms. Phylogenetic analyses indicate that the unique features of Halocynthia larval TnI likely represent derived features, and hence that the vertebrate-skeletal-muscle-like TnI of Ciona is a closer reflection of the ancestral ascidian larval TnI. Our results indicate that characteristics of vertebrate skeletal muscle TnI emerged early in the evolution of chordate locomotory muscle, before the ascidian/vertebrate divergence. These features could be related to a basal chordate locomotory innovation-e.g., swimming by oscillation of an internal notochord skeleton-or they may be of even greater antiquity within the deuterostomes.

Research paper thumbnail of Tissue-specific Alternative Splicing of Ascidian Troponin I Isoforms. REDESIGN OF A PROTEIN ISOFORM-GENERATING MECHANISM DURING CHORDATE EVOLUTION

Journal of Biological Chemistry, 1997

Research paper thumbnail of mRNA 5'-leader trans-splicing in the chordates

Genes & Development, 2001

We report the discovery of mRNA 5′-leader trans-splicing (SL trans-splicing) in the chordates. In... more We report the discovery of mRNA 5′-leader trans-splicing (SL trans-splicing) in the chordates. In the ascidian protochordate Ciona intestinalis, the mRNAs of at least seven genes undergo trans-splicing of a 16-nucleotide 5′-leader apparently derived from a 46-nucleotide RNA that shares features with previously characterized splice donor SL RNAs. SLtrans-splicing was known previously to occur in several protist and metazoan phyla, however, this is the first report of SLtrans-splicing within the deuterostome division of the metazoa. SL trans-splicing is not known to occur in the vertebrates. However, because ascidians are primitive chordates related to vertebrate ancestors, our findings raise the possibility of ancestral SL trans-splicing in the vertebrate lineage.

Research paper thumbnail of Determinative properties of muscle lineages in ascidian embryos

Development, 1987

Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' a... more Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' as partial embryos often elaborate tissue-specific features typical of their constituent cell lineages. We used this property to study recent corrections of the ascidian larval muscle lineage and to compare the ways in which different lineages give rise to muscle. Our evaluation of muscle differentiation was based on histochemical localization and quantitative radiometric measurement of a muscle-specific acetylcholinesterase activity, and the development of myofilaments and myofibrils as observed by electron microscopy. Although the posterior-vegetal blastomeres (B4.1 pair) of the 8-cell embryo have long been believed to be the sole precursors of larval muscle, recent studies using horseradish peroxidase to mark cell lineages have shown that small numbers of muscle cells originate from the anterior-vegetal (A4.1) and posterior-animal (b4.2) blastomeres of this stage. Fully differentiated ...

Research paper thumbnail of The single MyoD family gene of Ciona intestinalis encodes two differentially expressed proteins: implications for the evolution of chordate muscle gene regulation

Development, 1997

A MyoD family gene was identified in the ascidian Ciona intestinalis and designated CiMDF (Ciona ... more A MyoD family gene was identified in the ascidian Ciona intestinalis and designated CiMDF (Ciona intestinalis Muscle Determination Factor). Expression of CiMDF was restricted to the muscle cells of the developing embryo and the body-wall muscle of adults. Northern blots showed that two differentially regulated CiMDF transcripts were expressed during development. A 1.8 kb transcript (CiMDFa) appeared first and was gradually replaced by a 2.7 kb transcript (CiMDFb). These transcripts encoded essentially identical MyoD family proteins with the exception of a 68 amino acid C-terminal sequence present in CiMDFb that was absent from CiMDFa. Although both CiMDFa and CiMDFb contained the cysteine-rich/basic-helix loop helix domain (Cys-rich/bHLH) present in all MyoD family proteins, only CiMDFb contained the region near the C terminus (Domain III) characteristic of this gene family. Genomic Southern blots showed that C. intestinalis has only one MyoD family gene, suggesting that CiMDFa and ...

Research paper thumbnail of Purification and characterization of an ascidian larval acerylcholinesterase

Biochimica et biophysica acta, 1980

Larval acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) of the ascidian Ciona int... more Larval acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) of the ascidian Ciona intestinalis (L.) was purified by a two-step affinity chromatography procedure. Concavanalin A-Sepharose chromatography in batches provided the initial purification and was followed by chromatography on columns to which competitive inhibitors of acetylcholinesterase had been attached. The most efficient of these used m-carboxyphenylmethylammonium iodide coupled to Sepharose 4B via a hydrophobic 6-carbon spacer. In combination with the concanavalin A-Sepharose step, this affinity resin yielded recoveries of 30-39% with specific activities ranging from 580-730 units/mg protein, a total purification of 5000-7000-fold. Analysis of this product by polycrylamide gel electrophoresis in the presence of SDS and beta-mercaptoethanol revealed a single major polypeptide of M(r) 65 000-70 000. This protein was identified as the basic catalytic subunit of acetylcholinesterase by its coelectrophoresis wit...

Research paper thumbnail of Determinative properties of muscle lineages in ascidian embryos

Development, 1987

Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' a... more Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' as partial embryos often elaborate tissue-specific features typical of their constituent cell lineages. We used this property to study recent corrections of the ascidian larval muscle lineage and to compare the ways in which different lineages give rise to muscle. Our evaluation of muscle differentiation was based on histochemical localization and quantitative radiometric measurement of a muscle-specific acetylcholinesterase activity, and the development of myofilaments and myofibrils as observed by electron microscopy. Although the posterior-vegetal blastomeres (B4.1 pair) of the 8-cell embryo have long been believed to be the sole precursors of larval muscle, recent studies using horseradish peroxidase to mark cell lineages have shown that small numbers of muscle cells originate from the anterior-vegetal (A4.1) and posterior-animal (b4.2) blastomeres of this stage. Fully differentiated ...

Research paper thumbnail of The single MyoD family gene of Ciona intestinalis encodes two differentially expressed proteins: implications for the evolution of chordate muscle gene regulation

A MyoD family gene was identified in the ascidian Ciona intestinalis and designated CiMDF (Ciona ... more A MyoD family gene was identified in the ascidian Ciona intestinalis and designated CiMDF (Ciona intestinalis Muscle Determination Factor). Expression of CiMDF was restricted to the muscle cells of the developing embryo and the body-wall muscle of adults. Northern blots showed that two differentially regulated CiMDF transcripts were expressed during development. A 1.8 kb transcript (CiMDFa) appeared first and was gradually replaced by a 2.7 kb transcript (CiMDFb). These transcripts encoded essentially identical MyoD family proteins with the exception of a 68 amino acid C-terminal sequence present in CiMDFb that was absent from CiMDFa. Although both CiMDFa and CiMDFb contained the cysteine-rich/basic-helix loop helix domain (Cys-rich/bHLH) present in all MyoD family proteins, only CiMDFb contained the region near the C terminus (Domain III) characteristic of this gene family. Genomic Southern blots showed that C. intestinalis has only one MyoD family gene, suggesting that CiMDFa and ...

Research paper thumbnail of Differentiation of tropomyosin-containing myofibrils in cleavage-arrested ascidian zygotes expressing acetylcholinesterase

Two muscle differentiation programs, acetylcholinesterase and tropomyosin-containing filaments an... more Two muscle differentiation programs, acetylcholinesterase and tropomyosin-containing filaments and fibrils, occur together in the same cleavage-arrested zygotes (1-celled) of the ascidian Ciona intestinalis. Coexpression in such undivided but developing 'embryos' is consistent with the idea that separate elements of muscle differentiation are related at some regulatory level, perhaps through a single multi-gene regulatory factor. Fertilized Ciona eggs were exposed to cytochalasin B for 20 h and then briefly reacted histochemically for acetylcholinesterase activity. Strongly reacting specimens were selected and processed for transmission electron microscopy to reveal regions of muscle ultrastructure. Every acetylcholinesterase-reactive zygote tested contained muscle contractile elements; no example lacking acetylcholinesterase was found with myofilaments and myofibrils. As demonstrated by immunogold labelling, a polyclonal antibody to tropomyosin from Ciona adult body wall re...

Research paper thumbnail of Regulation of One-Carbon Biosynthesis and Utilization in

The regulation of several enzymes involved in one-carbon metabolism was studied in a mutant of Es... more The regulation of several enzymes involved in one-carbon metabolism was studied in a mutant of Escherichia coli K-12 defective in S-adenosylmethionine synthetase. The mutant that was reported to have a low endogenous concentration of S-adenosylmethionine had elevated levels of N-5, 10-methylene tetrahydrofolate reductase and serine transhydroxymethylase, but the level of N-5, 10methylene tetrahydrofolate dehydrogenase was not altered. These results suggest that S-adenosylmethionine plays a role in the regulation of one-carbon production and utilization. An enzyme system that cleaved glycine to one-carbon units was demonstrated. The enzymes responsible for the cleavage of glycine were induced by exogenous glycine but were independent of S-adenosylmethionine or purine levels in the cell.

Research paper thumbnail of Two histospecific enzyme expressions in the same cleavage-arrested one-celled ascidian embryos

Fertilized eggs of the ascidian, Ciona intestinalis, were prevented from undergoing cytokinesis b... more Fertilized eggs of the ascidian, Ciona intestinalis, were prevented from undergoing cytokinesis but not nuclear division by treatment with cytochalasin B. After appropriate times, such cleavage-arrested multinucleate zygotes developed acetylcholinesterase of larval tail muscle and an alkaline phosphatase ordinarily localized in the larval endoderm tissues. Separate histochemical reactions on one of a pair of samples taken from the eggs of single animals provided examples (6134) in which the numbers of cytochalasin-treated embryos displaying the respective reaction product overlapped sufficiently (15-2996) to indicate that some of the zygotes had developed both enzymes in the same uncleaved single cell. With an actual dual-staining technique that can be applied to single cleavage-arrested zygotes, 62% of those developing a strong alkaline phosphatase reaction also had a strong acetylcholinesterase reaction. In other experiments, quantitative measurements of enzyme activity in homogenates of 114 single cleavage-arrested zygotes confirm directly that 18% of the zygotes produce both enzymes. There was no obligatory mutual exclusion of the potential for simultaneous expression of two tissue-specific characteristics that would ordinarily be segregated into different lineages during early cleavages. The cytoplasmic determinants believed responsible for these histotypic expressions can apparently function independently in the same cell.

Research paper thumbnail of Determinative properties of muscle lineages in ascidian embryos

Development (Cambridge, England), 1987

Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' a... more Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' as partial embryos often elaborate tissue-specific features typical of their constituent cell lineages. We used this property to study recent corrections of the ascidian larval muscle lineage and to compare the ways in which different lineages give rise to muscle. Our evaluation of muscle differentiation was based on histochemical localization and quantitative radiometric measurement of a muscle-specific acetylcholinesterase activity, and the development of myofilaments and myofibrils as observed by electron microscopy. Although the posterior-vegetal blastomeres (B4.1 pair) of the 8-cell embryo have long been believed to be the sole precursors of larval muscle, recent studies using horseradish peroxidase to mark cell lineages have shown that small numbers of muscle cells originate from the anterior-vegetal (A4.1) and posterior-animal (b4.2) blastomeres of this stage. Fully differentiated ...

Research paper thumbnail of Development of acetylchilinesterase during embryogenesis of the ascidian Ciona intestinalis

The Journal of experimental zoology, 1979

We have characterized the embryonic muscle cell cholinesterase of the solitary ascidian, Ciona in... more We have characterized the embryonic muscle cell cholinesterase of the solitary ascidian, Ciona intestinalis (L.). The effects of selective enzyme inhibitors and the inhibition of enzyme activity at high concentrations of substrate suggest that the muscle cell enzyme is an acetylcholinesterase (E.C. 3.1.1.7). After gastrulation and before hatching, acetylcholinesterase activity increased 35- to 40-fold; after hatching (18 hours postfertilization) this activity continued to increase, leveling off at about 36 hours of development. Histochemical observations showed that before hatching acetylcholinesterase was located principally in the muscle cells of the tail and, after hatching, it began to develop in cells of the adult musculature and brain. Inhibition of protein syntnesis by puromycin and of RNA synthesis by actinomycin D, suggest that both protein and RNA synthesis were required for the increase in acetylcholinesterase activity observed in unhatched embryos. Although the continued...

Research paper thumbnail of Messenger RNA regulation in human diploid fibroblasts

Journal of Cellular Physiology, 1977

Research paper thumbnail of Regulation of protein synthesis in human diploid fibroblasts: Reduced initiation efficiency in resting cultures

Journal of Cellular Physiology, 1978

Research paper thumbnail of Messenger RNA synthesis during early ascidian development

Developmental Biology, 1978

Research paper thumbnail of Lineage segregation and developmental autonomy in expression of functional muscle acetylcholinesterase mRNA in the ascidian embryo

Developmental Biology, 1984

Research paper thumbnail of Lineage segregation and developmental autonomy in expression of functional muscle acetylcholinesterase mRNA in the ascidian embryo

Developmental Biology, 1984

Research paper thumbnail of Purification and characterization of an ascidian larval acerylcholinesterase

Biochimica et biophysica acta, 1980

Larval acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) of the ascidian Ciona int... more Larval acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) of the ascidian Ciona intestinalis (L.) was purified by a two-step affinity chromatography procedure. Concavanalin A-Sepharose chromatography in batches provided the initial purification and was followed by chromatography on columns to which competitive inhibitors of acetylcholinesterase had been attached. The most efficient of these used m-carboxyphenylmethylammonium iodide coupled to Sepharose 4B via a hydrophobic 6-carbon spacer. In combination with the concanavalin A-Sepharose step, this affinity resin yielded recoveries of 30-39% with specific activities ranging from 580-730 units/mg protein, a total purification of 5000-7000-fold. Analysis of this product by polycrylamide gel electrophoresis in the presence of SDS and beta-mercaptoethanol revealed a single major polypeptide of M(r) 65 000-70 000. This protein was identified as the basic catalytic subunit of acetylcholinesterase by its coelectrophoresis wit...

Research paper thumbnail of Development of the Ascidian Embryo: Cell Fate Specification by Autonomous and Inductive Processes

... (D) The b7. B blastomeres also give rise to four undefined cells in the posterior, dorsal reg... more ... (D) The b7. B blastomeres also give rise to four undefined cells in the posterior, dorsal region of the tail (37). Page 290. ... Van Bene, den and Julin were the first to investigate the relationship between cells of the ascidian embryo and their later developmental fates (101). ...

Research paper thumbnail of Ascidian Larva Reveals Ancient Origin of Vertebrate-Skeletal-Muscle Troponin I Characteristics in Chordate Locomotory Muscle

Molecular Biology and Evolution, 2003

Ascidians are protochordates related to vertebrate ancestors. The ascidian larval tail, with its ... more Ascidians are protochordates related to vertebrate ancestors. The ascidian larval tail, with its notochord, dorsal nerve cord, and flanking rows of sarcomeric muscle cells, exhibits the basic chordate body plan. Molecular characterization of ascidian larval tail muscle may provide insight into molecular aspects of vertebrate skeletal muscle evolution. We report studies of the Ci-TnI gene of the ascidian Ciona intestinalis, which encodes the muscle contractile regulatory protein troponin I (TnI). Previous studies of a distantly related ascidian, Halocynthia roretzi, showed that different TnI genes were expressed in larval and adult muscles, the larval TnI isoforms having an unusual C-terminal truncation not seen in any vertebrate TnI. Here we show that, in contrast with Halocynthia, Ciona does not have a specialized larval TnI; the same TnI gene that is expressed in the heart and body-wall muscle of the sessile adult is also expressed in embryonic/ larval tail muscle cells. Moreover the TnI isoform produced in embryonic/larval muscle is identical to that produced in adult body-wall muscle, i.e., a 182-residue protein with the characteristic chain length and overall structure of vertebrate skeletal muscle TnI isoforms. Phylogenetic analyses indicate that the unique features of Halocynthia larval TnI likely represent derived features, and hence that the vertebrate-skeletal-muscle-like TnI of Ciona is a closer reflection of the ancestral ascidian larval TnI. Our results indicate that characteristics of vertebrate skeletal muscle TnI emerged early in the evolution of chordate locomotory muscle, before the ascidian/vertebrate divergence. These features could be related to a basal chordate locomotory innovation-e.g., swimming by oscillation of an internal notochord skeleton-or they may be of even greater antiquity within the deuterostomes.

Research paper thumbnail of Tissue-specific Alternative Splicing of Ascidian Troponin I Isoforms. REDESIGN OF A PROTEIN ISOFORM-GENERATING MECHANISM DURING CHORDATE EVOLUTION

Journal of Biological Chemistry, 1997

Research paper thumbnail of mRNA 5'-leader trans-splicing in the chordates

Genes & Development, 2001

We report the discovery of mRNA 5′-leader trans-splicing (SL trans-splicing) in the chordates. In... more We report the discovery of mRNA 5′-leader trans-splicing (SL trans-splicing) in the chordates. In the ascidian protochordate Ciona intestinalis, the mRNAs of at least seven genes undergo trans-splicing of a 16-nucleotide 5′-leader apparently derived from a 46-nucleotide RNA that shares features with previously characterized splice donor SL RNAs. SLtrans-splicing was known previously to occur in several protist and metazoan phyla, however, this is the first report of SLtrans-splicing within the deuterostome division of the metazoa. SL trans-splicing is not known to occur in the vertebrates. However, because ascidians are primitive chordates related to vertebrate ancestors, our findings raise the possibility of ancestral SL trans-splicing in the vertebrate lineage.

Research paper thumbnail of Determinative properties of muscle lineages in ascidian embryos

Development, 1987

Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' a... more Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' as partial embryos often elaborate tissue-specific features typical of their constituent cell lineages. We used this property to study recent corrections of the ascidian larval muscle lineage and to compare the ways in which different lineages give rise to muscle. Our evaluation of muscle differentiation was based on histochemical localization and quantitative radiometric measurement of a muscle-specific acetylcholinesterase activity, and the development of myofilaments and myofibrils as observed by electron microscopy. Although the posterior-vegetal blastomeres (B4.1 pair) of the 8-cell embryo have long been believed to be the sole precursors of larval muscle, recent studies using horseradish peroxidase to mark cell lineages have shown that small numbers of muscle cells originate from the anterior-vegetal (A4.1) and posterior-animal (b4.2) blastomeres of this stage. Fully differentiated ...

Research paper thumbnail of The single MyoD family gene of Ciona intestinalis encodes two differentially expressed proteins: implications for the evolution of chordate muscle gene regulation

Development, 1997

A MyoD family gene was identified in the ascidian Ciona intestinalis and designated CiMDF (Ciona ... more A MyoD family gene was identified in the ascidian Ciona intestinalis and designated CiMDF (Ciona intestinalis Muscle Determination Factor). Expression of CiMDF was restricted to the muscle cells of the developing embryo and the body-wall muscle of adults. Northern blots showed that two differentially regulated CiMDF transcripts were expressed during development. A 1.8 kb transcript (CiMDFa) appeared first and was gradually replaced by a 2.7 kb transcript (CiMDFb). These transcripts encoded essentially identical MyoD family proteins with the exception of a 68 amino acid C-terminal sequence present in CiMDFb that was absent from CiMDFa. Although both CiMDFa and CiMDFb contained the cysteine-rich/basic-helix loop helix domain (Cys-rich/bHLH) present in all MyoD family proteins, only CiMDFb contained the region near the C terminus (Domain III) characteristic of this gene family. Genomic Southern blots showed that C. intestinalis has only one MyoD family gene, suggesting that CiMDFa and ...

Research paper thumbnail of Purification and characterization of an ascidian larval acerylcholinesterase

Biochimica et biophysica acta, 1980

Larval acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) of the ascidian Ciona int... more Larval acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) of the ascidian Ciona intestinalis (L.) was purified by a two-step affinity chromatography procedure. Concavanalin A-Sepharose chromatography in batches provided the initial purification and was followed by chromatography on columns to which competitive inhibitors of acetylcholinesterase had been attached. The most efficient of these used m-carboxyphenylmethylammonium iodide coupled to Sepharose 4B via a hydrophobic 6-carbon spacer. In combination with the concanavalin A-Sepharose step, this affinity resin yielded recoveries of 30-39% with specific activities ranging from 580-730 units/mg protein, a total purification of 5000-7000-fold. Analysis of this product by polycrylamide gel electrophoresis in the presence of SDS and beta-mercaptoethanol revealed a single major polypeptide of M(r) 65 000-70 000. This protein was identified as the basic catalytic subunit of acetylcholinesterase by its coelectrophoresis wit...

Research paper thumbnail of Determinative properties of muscle lineages in ascidian embryos

Development, 1987

Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' a... more Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' as partial embryos often elaborate tissue-specific features typical of their constituent cell lineages. We used this property to study recent corrections of the ascidian larval muscle lineage and to compare the ways in which different lineages give rise to muscle. Our evaluation of muscle differentiation was based on histochemical localization and quantitative radiometric measurement of a muscle-specific acetylcholinesterase activity, and the development of myofilaments and myofibrils as observed by electron microscopy. Although the posterior-vegetal blastomeres (B4.1 pair) of the 8-cell embryo have long been believed to be the sole precursors of larval muscle, recent studies using horseradish peroxidase to mark cell lineages have shown that small numbers of muscle cells originate from the anterior-vegetal (A4.1) and posterior-animal (b4.2) blastomeres of this stage. Fully differentiated ...

Research paper thumbnail of The single MyoD family gene of Ciona intestinalis encodes two differentially expressed proteins: implications for the evolution of chordate muscle gene regulation

A MyoD family gene was identified in the ascidian Ciona intestinalis and designated CiMDF (Ciona ... more A MyoD family gene was identified in the ascidian Ciona intestinalis and designated CiMDF (Ciona intestinalis Muscle Determination Factor). Expression of CiMDF was restricted to the muscle cells of the developing embryo and the body-wall muscle of adults. Northern blots showed that two differentially regulated CiMDF transcripts were expressed during development. A 1.8 kb transcript (CiMDFa) appeared first and was gradually replaced by a 2.7 kb transcript (CiMDFb). These transcripts encoded essentially identical MyoD family proteins with the exception of a 68 amino acid C-terminal sequence present in CiMDFb that was absent from CiMDFa. Although both CiMDFa and CiMDFb contained the cysteine-rich/basic-helix loop helix domain (Cys-rich/bHLH) present in all MyoD family proteins, only CiMDFb contained the region near the C terminus (Domain III) characteristic of this gene family. Genomic Southern blots showed that C. intestinalis has only one MyoD family gene, suggesting that CiMDFa and ...

Research paper thumbnail of Differentiation of tropomyosin-containing myofibrils in cleavage-arrested ascidian zygotes expressing acetylcholinesterase

Two muscle differentiation programs, acetylcholinesterase and tropomyosin-containing filaments an... more Two muscle differentiation programs, acetylcholinesterase and tropomyosin-containing filaments and fibrils, occur together in the same cleavage-arrested zygotes (1-celled) of the ascidian Ciona intestinalis. Coexpression in such undivided but developing 'embryos' is consistent with the idea that separate elements of muscle differentiation are related at some regulatory level, perhaps through a single multi-gene regulatory factor. Fertilized Ciona eggs were exposed to cytochalasin B for 20 h and then briefly reacted histochemically for acetylcholinesterase activity. Strongly reacting specimens were selected and processed for transmission electron microscopy to reveal regions of muscle ultrastructure. Every acetylcholinesterase-reactive zygote tested contained muscle contractile elements; no example lacking acetylcholinesterase was found with myofilaments and myofibrils. As demonstrated by immunogold labelling, a polyclonal antibody to tropomyosin from Ciona adult body wall re...

Research paper thumbnail of Regulation of One-Carbon Biosynthesis and Utilization in

The regulation of several enzymes involved in one-carbon metabolism was studied in a mutant of Es... more The regulation of several enzymes involved in one-carbon metabolism was studied in a mutant of Escherichia coli K-12 defective in S-adenosylmethionine synthetase. The mutant that was reported to have a low endogenous concentration of S-adenosylmethionine had elevated levels of N-5, 10-methylene tetrahydrofolate reductase and serine transhydroxymethylase, but the level of N-5, 10methylene tetrahydrofolate dehydrogenase was not altered. These results suggest that S-adenosylmethionine plays a role in the regulation of one-carbon production and utilization. An enzyme system that cleaved glycine to one-carbon units was demonstrated. The enzymes responsible for the cleavage of glycine were induced by exogenous glycine but were independent of S-adenosylmethionine or purine levels in the cell.

Research paper thumbnail of Two histospecific enzyme expressions in the same cleavage-arrested one-celled ascidian embryos

Fertilized eggs of the ascidian, Ciona intestinalis, were prevented from undergoing cytokinesis b... more Fertilized eggs of the ascidian, Ciona intestinalis, were prevented from undergoing cytokinesis but not nuclear division by treatment with cytochalasin B. After appropriate times, such cleavage-arrested multinucleate zygotes developed acetylcholinesterase of larval tail muscle and an alkaline phosphatase ordinarily localized in the larval endoderm tissues. Separate histochemical reactions on one of a pair of samples taken from the eggs of single animals provided examples (6134) in which the numbers of cytochalasin-treated embryos displaying the respective reaction product overlapped sufficiently (15-2996) to indicate that some of the zygotes had developed both enzymes in the same uncleaved single cell. With an actual dual-staining technique that can be applied to single cleavage-arrested zygotes, 62% of those developing a strong alkaline phosphatase reaction also had a strong acetylcholinesterase reaction. In other experiments, quantitative measurements of enzyme activity in homogenates of 114 single cleavage-arrested zygotes confirm directly that 18% of the zygotes produce both enzymes. There was no obligatory mutual exclusion of the potential for simultaneous expression of two tissue-specific characteristics that would ordinarily be segregated into different lineages during early cleavages. The cytoplasmic determinants believed responsible for these histotypic expressions can apparently function independently in the same cell.

Research paper thumbnail of Determinative properties of muscle lineages in ascidian embryos

Development (Cambridge, England), 1987

Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' a... more Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' as partial embryos often elaborate tissue-specific features typical of their constituent cell lineages. We used this property to study recent corrections of the ascidian larval muscle lineage and to compare the ways in which different lineages give rise to muscle. Our evaluation of muscle differentiation was based on histochemical localization and quantitative radiometric measurement of a muscle-specific acetylcholinesterase activity, and the development of myofilaments and myofibrils as observed by electron microscopy. Although the posterior-vegetal blastomeres (B4.1 pair) of the 8-cell embryo have long been believed to be the sole precursors of larval muscle, recent studies using horseradish peroxidase to mark cell lineages have shown that small numbers of muscle cells originate from the anterior-vegetal (A4.1) and posterior-animal (b4.2) blastomeres of this stage. Fully differentiated ...

Research paper thumbnail of Development of acetylchilinesterase during embryogenesis of the ascidian Ciona intestinalis

The Journal of experimental zoology, 1979

We have characterized the embryonic muscle cell cholinesterase of the solitary ascidian, Ciona in... more We have characterized the embryonic muscle cell cholinesterase of the solitary ascidian, Ciona intestinalis (L.). The effects of selective enzyme inhibitors and the inhibition of enzyme activity at high concentrations of substrate suggest that the muscle cell enzyme is an acetylcholinesterase (E.C. 3.1.1.7). After gastrulation and before hatching, acetylcholinesterase activity increased 35- to 40-fold; after hatching (18 hours postfertilization) this activity continued to increase, leveling off at about 36 hours of development. Histochemical observations showed that before hatching acetylcholinesterase was located principally in the muscle cells of the tail and, after hatching, it began to develop in cells of the adult musculature and brain. Inhibition of protein syntnesis by puromycin and of RNA synthesis by actinomycin D, suggest that both protein and RNA synthesis were required for the increase in acetylcholinesterase activity observed in unhatched embryos. Although the continued...

Research paper thumbnail of Messenger RNA regulation in human diploid fibroblasts

Journal of Cellular Physiology, 1977

Research paper thumbnail of Regulation of protein synthesis in human diploid fibroblasts: Reduced initiation efficiency in resting cultures

Journal of Cellular Physiology, 1978

Research paper thumbnail of Messenger RNA synthesis during early ascidian development

Developmental Biology, 1978

Research paper thumbnail of Lineage segregation and developmental autonomy in expression of functional muscle acetylcholinesterase mRNA in the ascidian embryo

Developmental Biology, 1984

Research paper thumbnail of Lineage segregation and developmental autonomy in expression of functional muscle acetylcholinesterase mRNA in the ascidian embryo

Developmental Biology, 1984

Research paper thumbnail of Purification and characterization of an ascidian larval acerylcholinesterase

Biochimica et biophysica acta, 1980

Larval acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) of the ascidian Ciona int... more Larval acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) of the ascidian Ciona intestinalis (L.) was purified by a two-step affinity chromatography procedure. Concavanalin A-Sepharose chromatography in batches provided the initial purification and was followed by chromatography on columns to which competitive inhibitors of acetylcholinesterase had been attached. The most efficient of these used m-carboxyphenylmethylammonium iodide coupled to Sepharose 4B via a hydrophobic 6-carbon spacer. In combination with the concanavalin A-Sepharose step, this affinity resin yielded recoveries of 30-39% with specific activities ranging from 580-730 units/mg protein, a total purification of 5000-7000-fold. Analysis of this product by polycrylamide gel electrophoresis in the presence of SDS and beta-mercaptoethanol revealed a single major polypeptide of M(r) 65 000-70 000. This protein was identified as the basic catalytic subunit of acetylcholinesterase by its coelectrophoresis wit...