Lens epithelial cell mRNA. III. Elevated expression of macrophage migration inhibitory factor mRNA in galactose cataracts.

A lens epithelial (LE) cell cDNA clone, designated Clone 156, was isolated from a mature rat LE cDNA library by methods of subtractive hybridization. The cDNA sequence of Clone 156 was 521 nucleotides in length, excluding the poly(T)-tail, and it encoded an open reading frame of 115 amino acids. The translated protein shared extensive sequence similarities with macrophage migration inhibitory factor (MIF) from mouse lens and human T-cell lymphocytes.

Lens epithelial cell mRNA, II. Expression of a mRNA encoding a lipid-binding protein in rat lens epithelial cells.

A lens epithelial (LE) cell cDNA clone, designated pLELBP, was isolated by subtraction-hybridization methods between a 4-week-old rat LE cell and rat lens fiber cell lambda ZAP cDNA libraries. The cDNA contained 683 bp, an ATG at bp 42, and an open reading frame (ORF) encoding a protein of 135 amino acids (aa), and a poly(A) signal at bp 641 (GenBank/EMBL accession No. U13253).

Lens epithelial cell mRNA. I. Cloning and sequencing of a messenger RNA with a basic motif/leucine-rich domain specifically expressed in rat lens epithelial cells.

By methods of subtraction-hybridization of lambda ZAP cDNA libraries, prepared from 4-week-old rat lens epithelial cells (capsule) and lens fiber cells (decapsulated lens), we have isolated a specific cDNA clone whose target mRNA is about 600 b long. Northern blot hybridization analysis data showed that the target mRNA was preferentially expressed in the lens epithelial cells; it was not found in the retina or in non-ocular tissues. The complete sequence of the mRNA was obtained both by the 5' and 3' rapid amplification of cDNA ends (5'-RACE, 3'-RACE), and by sequencing of a clone containing the full-length cDNA insert.

cDNA cloning, sequencing and in situ localization of a transcript specific to both sublingual demilune cells and parotid intercalated duct cells in mouse salivary glands.

A cDNA clone derived from mouse sublingual gland was isolated from lambda-phage cDNA library. Northern blot hybridization indicated that the transcript from which it was derived was approx. 700 nucleotides in length.

Levels of expression of the genes for glutathione reductase, glutathione peroxidase, catalase and CuZn-superoxide dismutase in rat lens and liver.

Oxidative mechanisms are thought to play a major role in several biological phenomena, including cataract formation. In the following studies we determined the relative levels of expression of the genes for the mRNAs for glutathione peroxidase (GPx), glutathione reductase (GR), CuZn-superoxide dismutase (CuZn-SOD) and catalase, in both the rat lens and liver. Northern blot hybridization methods were used to determine the mRNA size.

Galactose-induced cataract formation in guinea pigs: morphologic changes and accumulation of galactitol.

Purpose: To develop and characterize a new model of galactose-induced cataract formation in young, 3- to 4-week-old Hartley guinea pigs.

Methods: Experimental animals were fed 50% galactose in powdered guinea pig chow containing 0.5 g ascorbate/kg diet.

Abnormal expression of aldose reductase mRNA in fiber cells of cataractous rat lens. Analysis by in situ hybridization.

Aldose reductase (AR), an enzyme of the polyol pathway, has been implicated in the pathogenesis of diabetic and galactosemic cataracts. AR mRNA is a specific transcript of the lens epithelial cells. However, in addition to its presence at high levels in the epithelial cells at the equator, it is also found at significant concentrations in fiber cells at the lens bow.

Sorbitol dehydrogenase. Full-length cDNA sequencing reveals a mRNA coding for a protein containing an additional 42 amino acids at the N-terminal end.

A cDNA clone encoding rat sorbitol dehydrogenase (SDH) was isolated from a rat testis lambda ZAP II cDNA library. The full-length cDNA insert contained 2277 base pairs (bp), starting 182 bp upstream from an ATG codon where translation to the active enzyme SDH is presumed to be initiated. A second ATG codon, however, was found 126 bp upstream, aligned in the same reading frame as that of the active enzyme.

Levels of expression of hexokinase, aldose reductase and sorbitol dehydrogenase genes in lens of mouse and rat.

The level of expression of the genes for hexokinase, aldose reductase and sorbitol dehydrogenase was investigated in lenses of mice and rats. These genes represent two separate but interrelated pathways for the metabolism of glucose in the cell. It is hypothesized that the extent of expression of the hexokinase gene may play an important role in the regulation of the levels of glucose in the lens.

Profile of messenger RNA decay in the Emory mouse lens in cataractogenesis and in aging.

The Emory mouse is presumed to be a model for studies on human senile cataracts. The cataract develops in 5-8 months after birth, and it does not appear to have an osmotic component. To date, no specific metabolic lesion has been uncovered as a probable cause to this cataract.

Transient elevation of aldose reductase mRNA in lens of rats developing galactose cataracts.

Aldose reductase (AR), a major enzyme in the polyol pathway, is thought to be responsible for accumulation of polyols in lenses exposed to high doses of galactose or glucose, and it may be linked to some of the complications found in diabetes. In this report we examined the level of expression of AR mRNA in lens epithelia undergoing development of galactose cataracts in vivo. The AR mRNA was quantitated by Northern blot hybridization with a [35S]-RNA transcript from a previously described AR cDNA clone.

Expression of c-myc protooncogene in rat lens cells during development, maturation and reversal of galactose cataracts.

It is well established that normal patterns of epithelial cell proliferation and metabolism, and of fiber cell differentiation and maturation are essential for the maintenance of transparency in the ocular lens. Several factors, including exposure to high levels of sugars, have been known to result in the compromise of lens transparency. For example, initiation of lens cell damage by galactose induces lens epithelial cells to proliferate.

Crystallin mRNA concentrations and distribution in lens of normal and galactosemic rats. Implications in development of sugar cataracts.

It is well established that high concentrations of sugar in the lens of the eye eventually lead to fiber cell destruction and cataracts. In these studies the decrease in crystallin mRNAs was quantified as a result of influx of high concentrations of galactose into the lens of rats. The alpha A-, alpha B1-, and gamma-crystallin mRNA concentrations were assessed in normal lens and in lens undergoing development of sugar cataracts by northern blot and in situ hybridization methods.

Evaluation of lens epithelial cell differentiation by quantitation of MP26 mRNA relative to gamma-crystallin mRNA in initiation of galactose cataracts in the rat.

It is well established that in response to feeding of galactose to four-week-old rats cataracts develop (Unakar, Genyea, Reddan & Reddy, Exp. Eye Res. 26, 123-33, 1978).

Aldose reductase mRNA is an epithelial cell-specific gene transcript in both normal and cataractous rat lens.

Aldose reductase (AR) is implicated in the development of sugar cataracts by its reduction of galactose or glucose to polyols. The authors' recent work suggested that AR mRNA is found to be expressed in high concentrations in rat-lens epithelial cells after exposure of the animal to a diet containing 50% galactose. They localized the AR mRNA in the lens cells by in situ hybridization with a previously described AR clone.

Management of aldose reductase mRNA abundance in rat lens undergoing reversal of galactose induced cataracts. A model for gene response to changes in the environment.

Aldose reductase (AR) mRNA concentration in rat lens was quantitated by hybridization of a RNA transcript from a previously described AR cDNA clone to mRNA found in epithelial and cortical cytosols. This was done on normal rat lens and on lens initially made cataractous by feeding of a diet of Purina Chow containing 50% galactose, followed by reversal of the cataracts due to the removal of the galactose from the diet. Recent data from this laboratory has shown that AR mRNA was increased in lens epithelial cells upon administration of galactose; while in the cortex it was reduced to insignificant levels when fiber cell damage became extensive by day 20 on galactose.

Relative abundance of aldose reductase mRNA in rat lens undergoing development of osmotic cataracts.

Aldose reductase (AR) messenger RNA concentration was determined in normal rat lens and in lens from rats fed a 50% galactose diet over a period of 20 days. The AR mRNA was detected by using a previously described AR cDNA clone. The relative concentration of the AR mRNA was estimated by cpm of 35S-UTP labeled antisense RNA hybridized to dot-blots prepared from cytosols isolated from single lens, decapsulated lens (cortex) and its respective capsule (epithelia).

Messenger RNA population in normal and cataractous rat lens. A minireview.

Previous work from this laboratory has suggested that swollen nucleated fiber cells can survive in mature galactose-cataracts. Evidence for this observation was derived from analysis on the in vitro translation products of mRNA isolated from normal lens and lens undergoing development of galactose-cataracts. Additional studies on the abundance of a fiber cell specific gene product (MP26 mRNA) in both normal and cataractous lens mapped out gene response to: (1) differentiation of epithelial cells to fiber cells, (2) levels of this differential gene activity and its anatomical location in initiation and maturation of galactose-cataracts, and (3) distribution of MP26 mRNA in fibers of normal and cataractous lens.

MP26 messenger RNA sequences in normal and cataractous lens. A molecular probe for abundance and distribution of a fiber cell-specific gene product.

Previous work from this laboratory has suggested that swollen nucleated fiber cells can survive in mature galactose-cataracts. Evidence for this observation was derived from analysis on the in vitro translation products of mRNA isolated from normal lens and lens undergoing development of galactose-cataracts. Therefore, studies on the fate of a fiber cell-specific gene product (MP26 mRNA) in both normal and cataractous lens should map out gene response to: (1) differentiation of epithelial cells to fiber cells; (2) levels of this differential gene activity and its anatomical location in initiation and maturation of galactose-cataracts; and (3) distribution of MP26 and mRNA in fibers of normal and cataractous lens.

Crystallin mRNA product levels in lens undergoing reversal and inhibition of galactose cataracts.

In our previous work, two-dimensional gel electrophoretic analysis of the translational products from mRNA of lens from rats maintained on 50% galactose up to 45 days has suggested that synthesis of mRNA was not arrested by the disease process, but it decreased significantly relative to the control. The loss in mRNA number was due mainly to loss in cell population. Specifically, the gamma-crystallin mRNA product decreased to very low levels at onset of the disease.

Survival of fiber cells and fiber-cell messenger RNA in lens of rats maintained on a 50% galactose diet for 45 days.

Previous work from this laboratory has shown that in cataractous lens from rats fed a 50% galactose diet up to 32 days, synthesis of crystallin mRNAs was reduced while synthesis of mRNAs for the non-crystallin proteins appeared to be maintained at equivalent or higher levels than found in the controls. In this study, we find that the population of mRNA from the 45-day cataractous lens included all of the crystallin mRNAs and substantial amounts of mRNAs for the non-crystallin proteins, in particular those proteins of molecular weights ranging from 45,000 to 92,000, and pIs from 5.5 to 7.

Partial characterization of chromosomal proteins tightly bound to chicken erythroid DNA.

Extraction of chicken reticulocyte and erythrocyte chromatins with 2 M NaCl yields a small fraction (about 5%) of the total DNA which is very tightly bound to a class of nonhistone chromatin proteins (DNA-P). This DNA fraction has previously been shown to be significantly enriched in active gene sequences. The proteins associated with reticulocyte and erythrocyte DNA-P were analyzed by two-dimensional gel electrophoresis.

Total poly(A+)-messenger RNA from bovine lens cofractionates with sucrose purified fiber cell plasma membrane.

Poly(A+)-messenger RNA was isolated from bovine lens as well as poly(A+)-mRNA from crude and sucrose-purified plasma membrane. Bovine lens MP26 antisera was also propagated in rabbits, from which anti-MP26 IgG was partially purified and used to assay for the specific synthesis of MP26 during in vitro translation of the isolated poly(A+)-mRNAs. We found that membrane-associated poly(A+)-mRNA supported the synthesis of proteins which were identical to those translated from total lens fiber cell poly(A+)-mRNA.

Nuclear matrix DNA from chicken erythrocytes contains beta-globin gene sequences.

Nuclear matrices containing residual DNA were isolated from chicken erythrocytes after extraction of purified nuclei with buffered 2 M NaCl. After further purification of this residual DNA, it was found to contain high concentrations of beta-globin gene sequences as assayed by dot hybridization with 32P-labeled nick-translated pHB1001. Electron microscopy of a random sample of this residual DNA fraction shows the DNA to be intimately associated with protein at various intervals.

The effect of estrogen on the concentration of ovalbumin gene sequence in the 2 M NaCl residual fraction of oviduct chromatin.

Oviduct chromatin was isolated from both estrogenized and non-estrogenized hens. Extraction of the chromatin with 2 M NaCl removed a majority of the proteins, and the resulting DNA was then separated into two components: (1) a major fraction which was virtually protein-free; and (2) a minor fraction which was complexed with proteins. It was found that the DNA fraction that is complexed with proteins contained ovalbumin gene sequences and that the concentration of these sequences could be boosted by estrogen-treatment.