Crystallin synthesis by Chick Lens

By using the technique of laser light-scattering spectroscopy, direct observation has been made on the intracellular accumulation of a crystallin protein within the cells of chicken embryo lens during the process of development. Appearance of 8-crystallin has been detected as early as day 4, and its concentration reaches a plateau at day 19. The measurements constitute a noninvasive determination of accumulation of protein molecules that specifically characterize the process of cell differentiation.Rapid progress is being made in the understanding of molecular events of cellular differentiation, particularly at the level of gene expression. Lens cell differentiation has been a favorable system of investigation because it is associated with a marked change in morphology (cell elongation) and differential synthesis of the soluble proteins (crystallins). The crystallins, or structural proteins within the lens, comprise as much as 90% of the soluble protein of the lens. There are four families of crystallins (a-, f3, y, and 8-crystallin), with 6-crystallin being confined to birds and reptiles (1,2). Reviews on lens differentiation and lens crystallins can be found elsewhere (3-6).In the chicken lens, 8-crystallin is the first crystallin to appear (7) and can be detected already at the lens placode stage of development (8, 9). This protein accumulates in the embryonic lens until it comprises 70-80% of the soluble protein in the developing lens fiber cells (10-12). 8-Crystallin synthesis gradually ceases during the first few months after hatching (13)(14)(15). Consequently, the concentration of 8-crystallin is high in the center (nucleus) of the adult lens, which contains cells deposited early in development, and is low or absent at the periphery of the lens (cortex), which contains the cells deposited later in development (16) (Fig. 1) where q = (4ir/X) sin(6/2) is the scattering wave number, X is the wavelength of the laser light in water, 6 is the scattering angle (. = 900 in this experiment), and A and B are constants proportional to the square of concentration and molecular weight of the macromolecules. The diffusion coefficient is further related, via the Stokes-Einstein-Kawasaki-Ferrel formula, to the correlation length, a, of the macromolecules D = kT/6mrra, where k is the Boltzmann constant, T is the absolute temperature, and qj is the viscosity of water. The correlation length a is the average distance beyond which the motion of neighboring macromolecules becomes independent. When interaction is negligible, the correlation length is simply the hydrodynamic radius of the macromolecules.When there are macromolecules of different sizes and, therefore, different diffusion coefficients, the correlation function is given by the superposition of the correlation functions corresponding to each size. By precise analysis of the correlation function, it is, in principle, possible to determine the size distribution and the concentration of the macromolecules. 785The publication costs of this article were defray...

mentioning

confidence: 99%

Direct observation of delta-crystallin accumulation by laser light-scattering spectroscopy in the chicken embryo lens.

Sun¹,

Tanaka²,

Nishio³

et al. 1984

“…Chicken b-crystallin is a polymeric protein (6, 7) of about 200,000 daltons (8). The b-crystallin subunits have similar tryptic peptides and can be resolved by electrophoresis into two bands of 48,000 and 50,000 daltons (9, 10).Chicken 5-crystallin is particularly suited to developmental studies because it is the first crystallin to appear in the embryonic lens (3,11,12), and its rate and location of synthesis within the lens change during development (4,5,(11)(12)(13). Moreover, chicken lenses can be readily obtained from embryos at known stages of development, and lens cell differentiation involving 6-crystallin gene expression can be studied in tissue (5,11,14) and cell (15, 16) culture.…”

mentioning

confidence: 99%

Molecular cloning and partial characterization of delta-crystallin cDNA sequences in a bacterial plasmid.

Bhat¹,

Piatigorsky²

1979

Double-stranded cDNA synthesized from 5-crystallin mRNA isolated from lens fiber cells of 15-day-old embryonic chicken was cloned in Escherichia coli X1776 in the Pst I site of the plasmid pBR322 by using the oligo(dC);'oligo(dG) joining procedure. Twelve Amps Tetr transformants contained sequences complementary to purified 6-crystallin [32P]cDNA. One of the recombinant clones (pbCr-2) had an insert of 1241 + 240 base pairs, as judged by R-looping analysis with purified -crystallin mRNA. The inserted cDNA represents at least 69% of the 6-crystallin coding sequences. pbCr-2 was further characterized by restriction analysis, protection of -crystallin[3H]cDNA from digestion by SI nuclease, and hybrid-mediated arrest of -crystallin mRNA translation in vitro. p6Cr-2 provides an invaluable probe for additional analysis of the primary structure, gene organization, and regulated synthesis of &-crystallin, the principal protein synthesized during lens differentiation in the chicken embryo. The major constituents of the vertebrate lens are four classes of structural proteins termed crystallins (a, 3, y, and 3) (see refs. 1 and 2). By contrast to the other crystallins, 6-crystallin is present only in avian and reptilian lenses (see 2). 6-Crystallin has been most intensively studied in chickens, in which it represents 60-80% of the protein in the embryonic lens and about 50% of the protein in the adult lens (3-5). Chicken b-crystallin is a polymeric protein (6, 7) of about 200,000 daltons (8). The b-crystallin subunits have similar tryptic peptides and can be resolved by electrophoresis into two bands of 48,000 and 50,000 daltons (9, 10).Chicken 5-crystallin is particularly suited to developmental studies because it is the first crystallin to appear in the embryonic lens (3,11,12), and its rate and location of synthesis within the lens change during development (4,5,(11)(12)(13). Moreover, chicken lenses can be readily obtained from embryos at known stages of development, and lens cell differentiation involving 6-crystallin gene expression can be studied in tissue (5,11,14) and cell (15, 16) culture.In the present communication we have taken advantage of our ability to purify 6-crystallin mRNA (17,18) Construction of a Recombinant cDNAkpBR322 DNA. The restriction endonuclease Pst I was used to linearize the pBR322 DNA (Bethesda Research Laboratories, Rockville, MD). The linear plasmid DNA was tailed with oligo(dG) in the presence of 1 mM CoCl2 by terminal transferase (19), extracted with phenol, and precipitated with ethanol. The oligo(dG)-tailed plasmid DNA and the oligo(dC)-tailed ds cDNA were hybridized at 65°C in 10 mM Tris-HCl, pH 7.5/100 mM NaCl/1 mM EDTA for 2 hr. The reaction tubes were left at room temperature for 6 hr and then kept at 4°C until used to transform Escherichia coli X1776.Transformation of E. coli X1776 and Identification of Clones. All procedures were conducted in a P3 physical containment facility in compliance with National Institutes of Health Guidelines for Recombinant DNA Research. E. ...

“…b-Crystallin, a structural protein of the ocular lens, has several interesting features. First, b-crystallin is a useful marker for studying differential gene expression because it is the first (1)(2)(3) and principal (4)(5)(6) crystallin synthesized during lens development in the chicken. Second, b-crystallin is a tetrameric protein (7) composed of subunits with molecular weights of 50,000 and 48,000 (8).…”

mentioning

confidence: 99%

“…The cloned sequence does not represent a complete b-crystallin gene because approximately 500 nucleotides from the 3' end of the b-crystallin mRNA does not hybridize to this DNA (the unhybridized RNA tail has been broken during spreading and is not visible in the electron micrograph). Measurements of the intervening and mRNA gene sequences showed that regions D-I and [4][5][6][7][8][9][10] (Figs. 3 and 4) are the same as those shown for gbCr4 in Fig.…”

mentioning

confidence: 99%

Comparison of two delta-crystallin genes in the chicken.

Bhat¹,

Sullivan²,

Piatigorsky³

1980

Restriction endonuclease mapping and electron microscopy established that there are at least two nonalielic 5-crystallin genes in the chicken. The structure of a complete 5-crystallin gene has been deduced by analysis of five cloned fragments from the chicken genome. Fifteen intervening sequences compose approximately 80% of this gene; the other gene contains at least 14 intervening sequences. The mRNA gene sequences appear to be similar; at least 60% divergence was found between the intervening sequences of the two 5-crystallin genes. Insertions or deletions or both appear to be major events responsible for these differences. b-Crystallin, a structural protein of the ocular lens, has several interesting features. First, b-crystallin is a useful marker for studying differential gene expression because it is the first (1-3) and principal (4-6) crystallin synthesized during lens development in the chicken. Second, b-crystallin is a tetrameric protein (7) composed of subunits with molecular weights of 50,000 and 48,000 (8). It is not known whether the two subunits are products of different genes or whether one is posttranslationally derived from the other. The b-crystallin polypeptides are similar (8, 9): only two tryptic peptide differences have been detected between the 50,000 and 48,000 b-crystallin subunits (10). Interestingly, the ratio of synthesis of the subunits is a function of electrolyte concentrations in the cultured embryonic lens (11) or in a rabbit reticulocyte lysate supplemented with b-crystallin mRNA (12). Finally, 6-crystallin differs evolutionarily from the other lens crystallins in that it is found only in birds and reptiles (13,14).Recently, we have cloned a b-crystallin cDNA (pSCr2), allowing direct examination of b-crystallin sequences in the genome (15). Initial studies utilizing the cloned cDNA as a probe provided evidence that there are at least two 3-crystallin genes containing many intervening sequences in the chicken (16). In the present experiments we have examined five cloned fragments containing major portions of the b-crystallin genes by restriction endonuclease mapping and heteroduplex analysis.MATERIALS AND METHODS Preparation of DNA. Fertilized eggs from two lines of inbred White Leghorn chickens were obtained: one was gsnegative, Cofal Marek-negative (Spafas, Lancaster, PA) and one was line 6, subline 3 from the U.S. Department of Agriculture (Lansing, MI). DNA was prepared from pooled embryos of 6-to 12-day-old chickens by homogenizing 1 g of tissue in 10 vol of buffer A (0.1 M sucrose/3 mM CaCl2/3 mM Mg9l2/0.01 M Tris-HCl, pH 8.0/0.1% Triton X-100/0.5 mM dithiothreitol) (17). The homogenate was layered over a cushion of buffer A containing 0.3 M sucrose (without Triton X-100) and centrifuged at 3000 rpm in a Sorvall HB4 rotor. The nuclear pellet was resuspended in 100 vol of 0.01 M Tris-HCl, pH 8.0/0.15 M NaCl/0.01 M Na4EDTA, adjusted to 0.5% in NaDodSO4, and digested with 200 ,ug of proteinase K (Boehringer Mannheim) per ml at 370C for 15 hr. The DNA was then phenol ...