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We collected test reactions which can be applied in an easy and reproducible way to the chemical composition of the abalone organic matrix. Several chemical and biochemical test reactions were applied to the interlamellar organic matrix of abalone nacre to study the content and nature of polysaccharides. The preparation of the polysaccharide matrix was examined in parallel by light microscopy. The polysaccharide core is covered by a honeycomb-like structure, which can be completely removed by the protease subtilisin under release of the hydrophobic amino acid proline. The honeycomb pattern is in its size and its shape exactly the inverse matrix of the aragonite tablets, which are well-known to build up the nacreous layer of abalone shells. With this protocol we proved and verified in a straightforward and simple way the polysaccharides in abalone to be chitin. In addition, ¹H and ¹³C NMR analysis of the interlamellar organic matrix of abalone nacre confirmed that it consists to a high extent of the polysaccharide chitosan or its partially/completely N-acetylated derivative chitin (β-(1→4)-2-acetamido-2-deoxy-d-glucose or N-acetyl-d-glucosamine).

Chondrocytes grown in suspension contain both type I and type II collagen mRNAs, yet synthesize only type II collagen. The inability of chondrocytes to synthesize the α2 subunit of type I collagen, α2(I), results from a severely reduced translation elongation rate (Bennett, V.D., and Adams, S.L. (1987) J. Biol. Chem.262, 14806–14814). Furthermore, the α2(I) collagen mRNAs from chondrocytes are translated inefficiently in vitroand appear slightly smaller than those from other cells (Focht, R.J., and Adams, S.L. (1984) Mol. Cell. Biol.4, 1843–1852). These observations suggest that the reduced translation elongation rate may be due to an intrinsic property of the mRNAs. In this report we demonstrate that the α2(I) collagen mRNAs from suspended chondrocytes are 120 bases shorter than those from other cells, and that the first 94 bases of the chondrocyte mRNAs differ from the corresponding region of the calvaria mRNAs. The unique 5′ end of the chondrocyte α2(I) collagen mRNAs accounts for their smaller size and may be responsible for the translation elongation defect. Interestingly, the α2(I) collagen mRNAs from chondrocytes grown in monolayer, rather than in suspension, no longer display the cartilage-specific 5′ end, suggesting that cell shape and/or adhesion may modulate the structure of the 5′ end of the chondrocyte α2(I) collagen mRNAs.

Very little is known about the mechanisms mediating longevities of mRNAs. As a means of identifying potential cis- and trans-acting elements which stabilize an individual mRNA, naturally occurring mutations that decreased stability of the normally long-lived globin mRNA were analyzed. Our previous studies demonstrated that a subset of mutations which allowed the translating ribosome to read through into the alpha 2-globin 3' nontranslated region (NTR) targeted the mutant mRNAs for accelerated turnover in erythroid cells but not in several nonerythroid cell lines (I. M. Weiss and S. A. Liebhaber, Mol. Cell. Biol. 14:8123-8132, 1994). These results suggested that translational readthrough interfered with some feature of the alpha 2-globin 3' NTR required for message stability in erythroid cells. To define the cis-acting sequences which comprise this erythroid cell-specific stability determinant, scanning mutagenesis was performed on the alpha 2-globin 3' NTR, and the stability of each mutant mRNA was examined during transient expression. Three cytidine-rich regions which are required for longevity of the alpha 2-globin mRNA were identified. However, in contrast to the readthrough mutations, base substitutions in these elements destabilize the message through a translation-independent mechanism. To account for these results, we propose that the cis-acting elements form a complex or determinant in the normal alpha 2-globin mRNA which protects the message from degradation in erythroid cells. Disruption of this determinant, by translational readthrough or because mutations in an element prevent or inhibit its formation, targets the message for accelerated turnover in these cells.

The highly stable nature of globin mRNA is of central importance to erythroid cell differentiation. We have previously identified cytidine-rich (C-rich) segments in the human alpha-globin mRNA 3' untranslated region (alpha-3'UTR) which are critical in the maintenance of mRNA stability in transfected erythroid cells. In the present studies, we have detected trans-acting factors which interact with these cis elements to mediate this stabilizing function. A sequence-specific ribonucleoprotein (RNP) complex is assembled after incubation of the alpha-3'UTR with a variety of cytosolic extracts. This so-called alpha-complex is sequence specific and is not formed on the 3'UTR of either beta-globin or growth hormone mRNAs. Furthermore, base substitutions within the C-rich stretches which destabilize alpha-globin mRNA in vivo result in a parallel disruption of the alpha-complex in vitro. Competition studies with a series of homoribopolymers reveals a striking sensitivity of alpha-complex formation to poly(C), suggesting the presence of a poly(C)-binding activity within the alpha-complex. Three predominant proteins are isolated by alpha-3'UTR affinity chromatography. One of these binds directly to poly(C). This cytosolic poly(C)-binding protein is distinct from previously described nuclear poly(C)-binding heterogeneous nuclear RNPs and is necessary but not sufficient for alpha-complex formation. These data suggest that a messenger RNP complex formed by interaction of defined segments within the alpha-3'UTR with a limited number of cytosolic proteins, including a potentially novel poly(C)-binding protein, is of functional importance in establishing high-level stability of alpha-globin mRNA.

Although globin mRNAs are considered prototypes of highly stable messages, the mechanisms responsible for their longevity remain largely undefined. As an initial step in identifying potential cis-acting elements or structures which contribute to their stability, we analyzed the defect in expression of a naturally occurring alpha 2-globin mutant, alpha Constant Spring (CS). The CS mutation is a single-base change in the translation termination codon (UAA-->CAA) that allows the ribosome to read through into the 3' nontranslated region (NTR). The presence of CS mRNA in transcriptionally active erythroid precursors and its absence (relative to normal alpha-globin mRNA) in the more differentiated transcriptionally silent erythrocytes suggest that this mutation disrupts some feature of the alpha-globin mRNA required for its stability. Using a transient transfection system, we demonstrate that in murine erythroleukemia cells the CS mRNA is unstable compared with the normal alpha 2-globin mRNA. The analyses of several other naturally occurring and site-directed mutant alpha-globin genes in murine erythroleukemia cells indicate that entry of a translating ribosome into the 3' NTR targets the message for accelerated degradation in erythroid cells. In contrast, both the CS and alpha 2-globin mRNAs are stable in several nonerythroid cell lines. These results suggest that translational readthrough disrupts a determinant associated with the alpha 2-globin 3' NTR which is required for mRNA stability in erythroid cells.