Katherine Peterson

The determined state of white expression in the Drosophila eye is modified by zeste1 in the wzm family of mutants

Analysis of the whitezeste mottled (wzm) mutant family suggests that the zeste gene product functions in establishing and stabilizing a transcriptionally active chromatin domain for white locus expression. The z1 mutation reduces expression of paired or proximate copies of white, while single or unpaired copies maintain wildtype levels of expression. The wzmmutation, caused by the insertion of the retrotransposon BEL into the 5′ intron of white, alters the zeste-white interaction to produce a mottled eye phenotype in hemizygous z1wzm males. We have determined the molecular structure of four wzmderivatives. wzlresults from the insertion of an additional transposable element into the 5′ regulatory region of white. wzvlis a deletion of sequences upstream of the white locus. Two others, whaloand wcres, result from the transposition of wzmplus the entire verticals-roughest region into heterochromatin near the tip of chromosome 3L. They variegate for roughest but not for white; rather, the z1 effect on wzmnow causes white expression to become non-autonomous and non-clonal. The analysis of these five mutations shows that the neomorphic zeste1 product, in combination with structural changes imposed by transposons and intercalary heterochromatin, modifies the determination and stability of white expression. We propose that the normal zeste product functions as part of a complex that stimulates transcription by changing chromatin conformation to establish and maintain transcriptionally active domains. The unpairing of homologs is proposed to be one of the initial results of conformational change, providing an explanation for the role of zeste in transvection.

Expressed sequence tag analysis of adult human optic nerve for NEIBank: Identification of cell type and tissue markers

BackgroundThe optic nerve is a pure white matter central nervous system (CNS) tract with an isolated blood supply, and is widely used in physiological studies of white matter response to various insults. We examined the gene expression profile of human optic nerve (ON) and, through the NEIBANK online resource, to provide a resource of sequenced verified cDNA clones. An un-normalized cDNA library was constructed from pooled human ON tissues and was used in expressed sequence tag (EST) analysis. Location of an abundant oligodendrocyte marker was examined by immunofluorescence. Quantitative real time polymerase chain reaction (qRT-PCR) and Western analysis were used to compare levels of expression for key calcium channel protein genes and protein product in primate and rodent ON.ResultsOur analyses revealed a profile similar in many respects to other white matter related tissues, but significantly different from previously available ON cDNA libraries. The previous libraries were found to include specific markers for other eye tissues, suggesting contamination. Immune/inflammatory markers were abundant in the new ON library. The oligodendrocyte marker QKI was abundant at the EST level. Immunofluorescence revealed that this protein is a useful oligodendrocyte cell-type marker in rodent and primate ONs. L-type calcium channel EST abundance was found to be particularly low. A qRT-PCR-based comparative mammalian species analysis reveals that L-type calcium channel expression levels are significantly lower in primate than in rodent ON, which may help account for the class-specific difference in responsiveness to calcium channel blocking agents. Several known eye disease genes are abundantly expressed in ON. Many genes associated with normal axonal function, mRNAs associated with axonal transport, inflammation and neuroprotection are observed.ConclusionWe conclude that the new cDNA library is a faithful representation of human ON and EST data provide an initial overview of gene expression patterns in this tissue. The data provide clues for tissue-specific and species-specific properties of human ON that will help in design of therapeutic models.

Serum starvation of ARPE-19 changes the cellular distribution of cholesterol and Fibulin3 in patterns reminiscent of age-related macular degeneration

ABSTRACT Retinal pigment epithelium (RPE) has been implicated as key source of cholesterol‐rich deposits at Bruchs membrane (BrM) and in drusen in aging human eye. We have shown that serum‐deprivation of confluent RPE cells is associated with upregulation of cholesterol synthesis and accumulation of unesterified cholesterol (UC). Here we investigate the cellular processes involved in this response. We compared the distribution and localization of UC and esterified cholesterol (EC); the age‐related macular degeneration (AMD) associated EFEMP1/Fibulin3 (Fib3); and levels of acyl‐coenzyme A (CoA): cholesterol acyltransferases (ACAT) ACAT1, ACAT2 and Apolipoprotein B (ApoB) in ARPE‐19 cells cultured in serum‐supplemented and serum‐free media. The results were compared with distributions of these lipids and proteins in human donor eyes with AMD. Serum deprivation of ARPE‐19 was associated with increased formation of FM dye‐positive membrane vesicles, many of which co‐labeled for UC. Additionally, UC colocalized with Fib3 in distinct granules. By day 5, serum‐deprived cells grown on transwells secreted Fib3 basally into the matrix. While mRNA and protein levels of ACTA1 were constant over several days of serum‐deprivation, ACAT2 levels increased significantly after serum‐deprivation, suggesting increased formation of EC. The lower levels of intracellular EC observed under serum‐deprivation were associated with increased formation and secretion of ApoB. The responses to serum‐deprivation in RPE‐derived cells: accumulation and secretion of lipids, lipoproteins, and Fib3 are very similar to patterns seen in human donor eyes with AMD and suggest that this model mimics processes relevant to disease progression. HIGHLIGHTSSerum deprivation regulates cholesterol levels in confluent RPE cells.Serum deprivation leads to specific distributions of cholesterol and Fibulin3.The patterns of protein and lipid deposition resemble those in AMD eyes.

γ‐Crystallins of the chicken lens: remnants of an ancient vertebrate gene family in birds

γ‐Crystallins, abundant proteins of vertebrate lenses, were thought to be absent from birds. However, bird genomes contain well‐conserved genes for γS‐ and γN‐crystallins. Although expressed sequence tag analysis of chicken eye found no transcripts for these genes, RT‐PCR detected spliced transcripts for both genes in chicken lens, with lower levels in cornea and retina/retinal pigment epithelium. The level of mRNA for γS in chicken lens was relatively very low even though the chicken crygs gene promoter had lens‐preferred activity similar to that of mouse. Chicken γS was detected by a peptide antibody in lens, but not in other ocular tissues. Low levels of γS and γN proteins were detected in chicken lens by shotgun mass spectroscopy. Water‐soluble and water‐insoluble lens fractions were analyzed and 1934 proteins (< 1% false discovery rate) were detected, increasing the known chicken lens proteome 30‐fold. Although chicken γS is well conserved in protein sequence, it has one notable difference in leucine 16, replacing a surface glutamine conserved in other γ‐crystallins, possibly affecting solubility. However, L16 and engineered Q16 versions were both highly soluble and had indistinguishable circular dichroism, tryptophan fluorescence and heat stability (melting temperature Tm ~ 65 °C) profiles. L16 has been present in birds for over 100 million years and may have been adopted for a specific protein interaction in the bird lens. However, evolution has clearly reduced or eliminated expression of ancestral γ‐crystallins in bird lenses. The conservation of genes for γS‐ and γN‐crystallins suggests they may have been preserved for reasons unrelated to the bulk properties of the lens.

The klotho-related protein KLPH (lctl) has preferred expression in lens and is essential for expression of clic5 and normal lens suture formation

&NA; KLPH/lctl belongs to the Klotho family of proteins. Expressed sequence tag analyses unexpectedly revealed that KLPH is highly expressed in the eye lens while northern blots showed that expression is much higher in the eye than in other tissues. In situ hybridization in mouse localized mRNA to the lens, particularly in the equatorial epithelium. Immunofluorescence detected KLPH in lens epithelial cells with highest levels in the germinative/differentiation zone. The gene for KLPH in mouse was deleted by homologous recombination. Littermate knockout (KO) and wild type (WT) mice were compared in a wide panel of pathology examinations and were all grossly normal, showing no systemic effects of the deletion. However, the lens, while superficially normal at young ages, had focusing defects and exhibited age‐related cortical cataract by slit lamp examination. Whole‐lens imaging showed that KO mice had disorganized lens sutures, forming a loose double‐y or x instead of the tight y formation of WT. RNA‐seq profiles for KO and WT littermates confirmed the absence of KLPH mRNA in KO lens and also showed complete absence of transcripts for Clic5, a protein associated with cilium/basal body related auditory defects in a mouse model. Immunofluorescence of lens epithelial flat mounts showed that Clic5 localized to cilia/centrosomes. Mice mutant for Clic5 (jitterbug) also had defective sutures. These results suggest that KLPH is required for lens‐specific expression of Clic5 and that Clic5 has an important role in the machinery that controls lens fiber cell extension and organization. HighlightsKLPH has preferred expression in lens.Gene Deletion of KLPH produces lens suture defects.KLPH is essential for expression of Clic5 in lens.Clic5 locates to cilia/centrosomes in lens.Clic5 mutant mice have suture defects.