John Papaconstantinou

Stabilization of mRNA templates in bovine lens epithelial cells

Abstract Through the use of actinomycin D, it has been shown that the half-life of α-, β- and γ-crystallin mRNA in bovine lens epithelium increases with lens age. Crystallin synthesis in epithelial cells from embryonic lenses was potently inhibited by actinomycin D; the synthesis of these same proteins in adult lens epithelial cells was not affected. In both cases, all RNA synthesis was completely turned off by the antibiotic. Since the mitotic index of epithelial cells in embryonic lenses is much greater than that of the adult epithelial cells, our results indicate that stabilization of the protein-synthesizing templates found in adult epithelial cells may be related to the decreased replicative activity of these cells.

A change in the stoichiometry of assembly of bovine lens α-crystallin subunits in relation to cellular differentiation*

Summary Incorporation of [ 3 H] leucine into oligomeric α -crystallin via individual subunits has been measured in epithelial cells and cortex fiber cells from adult bovine lenses in vitro. Our data show that the ratio of [ 3 H] leucine incorporation via subunits α B 2 and α A 2 is shifted from a value of about 1:2 in epithelial cells to a value of about 1:3 in fiber cells. Thus, in this system, cellular differentiation is accompanied by a change in the stoichiometry of assembly of individual subunits to form the oligomeric α -crystallin molecule. These results indicate possible changes in the rates of synthesis of individual α -crystallin subunits.

A change in α-crystallin subunit composition in relation to cellular differentiation in adult bovine lens

Abstract The vertebrate lens is composed of two distinct cell types, the epithelial cell and the fiber cell. α-Crystallin, a structural protein found in both cell types, is composed of four subunits (αA 1 , αA 2 , αB 1 , and αB 2 ). In the epithelial cells the α-crystallin consists mainly of αA 2 and αB 2 with trace amounts of αA 1 and αB 1 . In the fiber cell there is a large increase in the amount of αA 1 and αB 1 subunits. This quantitative increase in two specific subunits is related to the process of cellular growth and differentiation in the vertebrate lens.

Increase in the complexity of alpha-crystallins during differentiation of lens cells.

PRELIMINARY studies of the changes in crystallin proteins occurring during development of the lens have indicated that the α-crystallins of foetal, calf and adult lenses differ in their chromatographic properties when fractionated on DEAE-cellulose1. The studies reported here indicate that the types of subunits present in bovine α-crystallins change during lens development. In a previous study, we showed that the α-crystallins can be resolved into five components by DEAE-cellulose chromatography, and that these multiple forms of α-crystallin contain identical subunit chains2. As they vary only in their proportions of these subunits, we have combined the five α-crystallin components into one fraction in the following experiments in order to get an overall picture of the types of α-crystallin subunits produced at different stages of development.

Glucocorticoids inhibit the coordinated translation of α- and β-globin mRNAs in friend erythroleukemia cells

Abstract The dimethylsulfoxide (Me 2 SO)-mediated induction of hemoglobin synthesis in Friend erythroleukemia cells is inhibited by the glucocorticoids hydrocortisone, dexamethasone, and fluocinolone acetonide; hydrocortisone, at concentrations of 10 −5 to 10 −8 m inhibits by 90−30% and fluocinolone acetonide at concentrations of 10 −8 to 10 −11 m shows a greater than 90% inhibition. At these concentrations the hormones have no effect on cell growth or viability. In this study it has been shown that there is a group of proteins, including the a- and β-globins, whose regulation is associated with the induction of Friend erythroleukemia cell differentiation, and that the expression of some of these, in addition to α- and β-globin, is affected by glucocorticoids. The levels of α- and β-globin mRNAs are very close to fully induced levels and preclude transcription as a major site for glucocorticoid control. In addition, it has been shown that glucocorticoids inhibit the translation of α- and β-globin mRNAs, that the level of this inhibition is concentration dependent, and that the translation of β-globin mRNA is slightly more sensitive to inhibition than the translation of α-globin mRNA. It is concluded that, although the translation of α- and β-globin mRNA is a major site of inhibition by glucocorticoids, there is a detectable amount of α- and β-globin synthesized. Thus, part of this mechanism may involve a differential sensitivity of α- and β-globin mRNA translation which results in unequal amounts of globin synthesis and an overall more potent inhibition of hemoglobin formation.

Molecular mechanism of extinction of liver-specific functions in mouse hepatoma x rat fibroblast hybrids: extinction of the albumin gene.

Hybrids formed by the fusion of mouse hepatoma (BWTG3) and rat fibroblast (JF1) cells exhibit the extinction of mouse albumin and α-fetoprotein synthesis. Karyotype analyses suggest that all parental chromosomes are present in the hybrids. The extinction, therefore, of mouse hepatocyte genes is attributed to the inhibitory action of the rat genome. In these studies, we show that these hybrids possess and express the mouse β-glucuronidase gene (which is encoded on the same chromosome as the mouse albumin and α-fetoprotein gene), and we present data of Southern blot analysis which demonstrate that such hybrids have indeed retained both mouse and rat albumin DNA sequences. In addition, using mouse albumin cDNA, we have shown by cDNA-RNA reassociation kinetics that albumin mRNA is virtually absent in these hybrids. We conclude from these studies that the extinction of albumin synthesis involves a mechanism which results in the loss of cytoplasmic albumin mRNA.

Synthesis of mitochondrial DNA in spermatocytes of Rhynchosciara hollaenderi

Abstract During the mid to late 4th instar period of larval development, the mitochondria of Rhynchosciara spermatocytes undergo highly characteristic morphological changes. In late meiosis the enlarged mitochondria fuse to form a single mitochondrial element which will ultimately extend the length of the spermatid tail. Our studies have shown that synthesis of a circular DNA occurs during this period of mitochondrial “differentiation.” This DNA has a density of 1.681 g/cm 3 ; and its synthesis cannot be detected in somatic tissues such as salivary gland, fat body, or gastric cecum. From analysis of DNA extracted from mitochondrial pellets, we have shown that the circular DNA is associated with the mitochondria. The contour length of the mitochondrial DNA is 9 μm, equivalent to a molecular weight of 18 × 10 6 . Although most metazoan mitochondrial DNAs exhibit contour lengths of approximately 5 μm (10 × 10 5 daltons), there is no extractable 5 μm circular DNA in these spermatocytes. Therefore, we conclude that either Rhynchosciara spermatocytes possess a distinct 9 μm mitochondrial DNA or that the spermatocyte mitochondrial DNA represents dimers of 5 μm monomers.

Differential incorporation of 5-bromodeoxyuridine into DNA puffs of larval salivary gland chromosomes in Rhynchosciara☆

Abstract An autoradiographic analysis of the incorporation of 5-bromo-2′-deoxyuridine (BUdR) into salivary gland chromosomes of Rhynchosciara larvae has demonstrated differential synthesis of DNA in a specific set of chromosomal puffs. DNA isolated from larval salivary glands at the specific stage in development when these DNA puffs occur has also been subjected to isopycnic centrifugation in CsCl. The profiles of these gradients are heterodisperse and exhibit regions of very high density shifted away from the bulk DNA. The technique makes possible the isolation and purification of sequences unique to a specific set of chromosomal loci.

Degradation of ribosomal RNA during chicken lens development

Abstract During the development of the vertebrate lens it acquires a low RNA content. Evidence indicates the persistence of a low molecular weight RNA while rRNA is depleted. This low molecular weight RNA has been characterized by chromatography, centrifugation and electrophoresis. Data indicate that it is primarily tRNA with small amounts of 5-S RNA. The persistence of the low molecular weight component is shown to be due to the selective retention of tRNA and not the accumulation of rRNA breakdown products. The degradation of rRNA is shown to occur on sedimentable ribonucleoprotein particles.

Generation and characterization of variants of mouse hepatoma cells with defects in hepato-specific gene expression. I. Albumin synthesis variants

Clonal variants of mouse hepatoma cells that either fail to produce albumin (variant 19/2) or show significantly reduced levels (100-fold less) of albumin production (variant 1/c/1) were isolated from the parental line, Hepa la, after a single exposure to N-methyl-N′-nitrosoguanidine (MNNG). Intracellular levels of albumin in both variants were below detection by our assay. Analyses by cDNA-RNA reassociation kinetics indicate that there are approximately 3900 molecules of cytoplasmic albumin mRNA per cell in the parent and less than 10 molecules per cell in both variants. Southern blotting of the Eco RI restriction fragments of cellular DNA from the parent and variants did not indicate any major deletions in the albumin gene DNA sequences. We conclude that in the two variants studied, processes that regulate albumin production via alterations in the level of cytoplasmic albumin mRNA have been affected. Our analyses have also shown that alpha-fetoprotein (AFP) production is lacking in one variant (19/2) and is slightly reduced in the other (1/c/1). Transferrin secretion is lower than the parental line in both variants. Thus multiple nonlethal defects in hepatic gene expression can be obtained in Hepa la cells in culture that will be useful in determining the number and kinds of genes that control the expression of liver-specific loci.