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C H Sullivan

Publications and source records attributed to C H Sullivan.

10 recordsLinked to original sources
PubMed1998

Reliability of delta-crystallin as a marker for studies of chick lens induction.

Induction of a lens by the optic vesicle of the brain was the first demonstration of how tissue interactions could influence cell fate during development. However, recent work with amphibians has shown that the optic vesicle is not the primary inducer of lens formation. Rather, an earlier interaction between anterior neural plate and presumptive lens ectoderm appears to direct lens formation. One problem with many early experiments was the absence of an unambiguous assay for lens formation. Before being able to test whether the revised model of lens induction applies to chicken embryos, we examined the suitability of using delta-crystallin as a marker of lens formation. Although delta-crystallin is the major protein synthesized in the chick lens, one or both of the two delta-crystallin genes found in chickens is transcribed in many non-lens tissues as well. In studies of lens formation where appearance of the delta-crystallin protein is used as a positive assay, synthesis of delta-crystallin outside of the lens could make experiments difficult to interpret. Therefore, polyacrylamide gel electrophoresis, immunoblotting, and immunofluorescence were used to determine whether the delta-crystallin messenger RNA detected in non-lens tissues is translated into protein, as it is in the lens. On Coomassie-blue-stained gels of several tissues from stage-22 embryos, a prominent protein was observed that co-migrated with delta-crystallin. However, on immunoblots, none of the nonlens tissues tested contained detectable levels of delta-crystallin at this stage. By imunofluorescence, delta-crystallin was observed in Rathke's pouch and in a large area of oral ectoderm near Rathke's pouch, yet none of the cells in these non-lens tissues showed the typical elongated morphology of lens fiber cells. When presumptive lens ectoderm or other regions of ectoderm from stage-10 embryos were cultured and tested for lens differentiation, both cell elongation and delta-crystallin synthesis were observed, or neither were observed. The results suggest that delta-crystallin synthesis and cell elongation together serve as useful criteria for assessing a positive lens response.

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PubMed1991

Delta-crystallin gene expression and patterns of hypomethylation demonstrate two levels of regulation for the delta-crystallin genes in embryonic chick tissues.

In this study we address two questions regarding the control of delta-crystallin gene expression in chick embryos. First we have determined whether delta-crystallin mRNA is found outside of the developing lens, in which it is the predominant mRNA. We find that this mRNA can be detected, although at relatively low levels, in all embryonic tissues we have examined (from the definitive streak stage onward). This low level of transcription may be related to a second function for one or both of the delta-crystallin genes: both genes have a high degree of sequence identity to the enzyme argininosuccinate lyase. This result led us to a second set of experiments in which we reevaluated the possible role of hypomethylation in the expression of the delta-crystallin genes. Previous work showed that particular HhaI and HpaII sites in the crystallin genes undergo hypomethylation early in the process of lens differentiation when there is a burst of delta-crystallin mRNA accumulation. We not find that these sites remain methylated in nonlens tissues, implying that they cannot be required for the delta-crystallin gene activity found in these tissues. Other sites are constitutively hypomethylated, however, and may be functionally linked to this low level of gene activity. From an analysis of the kinetics of the developmentally regulated hypomethylation of HhaI and HpaII sites we also find that complete hypomethylation of these sites is not required for activating high levels of delta-crystallin transcription during lens differentiation. We do find, however, that these sites approach a fully hypomethylated state later in the lens differentiation process. Our analyses of mRNA levels and hypomethylation together lead us to propose that the delta-crystallin genes are regulated by two different mechanisms, one that leads to high levels of expression in the lens and the other which is responsible for low level expression in all other tissues in the chick embryo.

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PubMed1989

Developmental regulation of hypomethylation of delta-crystallin genes in chicken embryo lens cells.

Sequences in the two delta-crystallin genes become hypomethylated when they are expressed in the chick lens. This system is particularly advantageous for studying temporal changes in hypomethylation, since lens tissue can be isolated at all developmental stages. In previous work we have shown that most HpaII sites become hypomethylated within the delta 1-crystallin gene long after delta-crystallin gene activation. One site is hypomethylated when crystallin mRNA begins to be synthesized at high levels at 50 h; we show here that this site maps to the 3' end (intron 15) of the delta 1-crystallin gene. In addition, we have examined the methylation status of HpaII and HhaI sites found near the 5' end of the delta 1-crystallin gene. Two HhaI sites adjacent to a viral core enhancer sequence in intron 2 are also first hypomethylated at 50 h. These findings point to regions of the delta 1 gene that should be investigated further for functional significance in regulating delta-crystallin transcription.

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PubMed1987

Delta-crystallin genes become hypomethylated in postmitotic lens cells during chicken development.

Although it has been argued that the loss of 5-methylcytosine from specific sites in DNA plays an important role in activation of specific genes, the mechanism of hypomethylation is not well understood. One model links the process to DNA replication, proposing that it occurs by not remethylating cytosine on newly synthesized DNA. An alternative model argues that hypomethylation results from excision of part or all of the 5-methylcytosine. We were able to test whether hypomethylation can occur without replication by analysis of methylation changes in the delta-crystallin genes of the chicken lens. During embryonic development a large fraction of cells in the lens stops dividing as part of the differentiation process. Shortly after this stage, the delta-crystallin genes in samples of the whole lens become hypomethylated, suggesting the possibility that this process might be occurring in the subset of cells that is no longer dividing. We found that hypomethylation of these genes does occur in postmitotic lens cells, a result that implicates an excision mechanism in this tissue.

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PubMed1982

Purification of xanthine oxidase from the fat-globule membrane of bovine milk by electrofocusing.

Xanthine oxidase was purified from bovine milk-fat globule membrane by extraction with butan-1-ol, precipitation with ammonium sulphate, separation by preparative electrofocusing and chromatography on Concanavalin-A/Agarose. The enzyme had an A280/A450 ratio of 4.8 and a specific activity of 3.09. At least five to seven variants of the enzyme with isoelectric points from pH 6.9 to 7.6 were identified. Previously identified minor "variants' of the enzymes with apparently acidic isoelectric points (1) were shown to be the result of aggregation of enzyme with membrane sialoglycoproteins. Specific antibodies to xanthine oxidase were prepared by fractionating immune serum on a column of enzyme covalently bound to Sepharose 4B. A single immunoprecipitate was obtained when the purified antibodies were allowed to diffuse in agarose gels against either Triton-X-100-extracted membrane or purified xanthine oxidase. Immunoelectrophoresis of the enzyme against anti-sera to xanthine oxidase, however, revealed two precipitin lines, both of which were positive when histochemically stained for enzyme activity. The results are discussed with reference to previous purification schemes for xanthine oxidase and previous estimates for the isoelectric points of the enzyme. We also outline practical uses for the antibody prepared against the enzyme in this present study.

Animals↗
PubMed1982

Detection of xanthine oxidase and immunologically related proteins in fractions from bovine mammary tissue and milk after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate.

A solid-phase immunoassay was used to detect xanthine oxidase in fractions from bovine mammary glands after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Under these conditions the major proportion of xanthine oxidase in either mammary tissue or mild could be recovered as a protein of mol.wt. 150 000. In mammary tissue approx. 80% of the enzyme was in a soluble form and the remainder was accounted for in either 'mitochondrial' or microsomal fractions after tissue homogenization and fractionation. Affinity chromatography of either detergent-solubilized microsomal membranes or postmicrosomal supernatants on immobilized antibody to xanthine oxidase yielded a single protein that cross-reacted with antibody to the enzyme. In milk presumptive degradation products of the enzyme were detected in minor quantities with mol.wts. of 43 000 in the whey fraction and 90 000 in fat-globule membrane. Only the undegraded enzyme was present in the skim-milk membrane fraction. Xanthine oxidase is therefore synthesized and secreted as a protein with a monomeric mol.wt. of 150 000 and is not subjected to extensive proteolytic degradation during the storage of milk in mammary alveoli. The significance of the results is discussed in relation to the overall protein composition of the membranes of milk-fat globules and skim milk.

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