Fujiko Watt

Prostate-specific suicide gene therapy using the prostate-specific membrane antigen promoter and enhancer

Prostate‐specific membrane antigen (PSMA) is abundantly expressed in virtually 100% of prostate cancers and metastases. In addition, unlike prostate‐specific antigen (PSA), PSMA is upregulated under conditions of androgen deprivation. Therefore, PSMA is an attractive therapeutic target for advanced prostate cancer. Recently, both the promoter and the enhancer driving prostate‐specific expression of the PSMA gene were cloned. We describe here our analysis of the PSMA enhancer for the most active region(s) and present a way of using the enhancer in combination with the E. coli cytosine deaminase gene for suicide‐driven gene therapy that converts the nontoxic prodrug 5‐fluorocytosine (5‐FC) into the cytotoxic drug 5‐fluorouracil (5‐FU) in prostate cancer cells.

Relative activity and specificity of promoters from prostate-expressed genes

To evaluate their relative activity and specificity for prostate cells promoter and regulatory regions from three prostate‐expressed genes–prostate‐specific antigen (PSA), probasin, and relaxin H2–have been compared in prostate cell lines and in lines of breast, bladder, liver, kidney, lung, and ovarian origin.

Chromosomal proteins of Drosophila melanogaster and an approach for their localisation on polytene chromosomes

Chromosomal proteins have been prepared from embryos of Drosophila melanogaster and separated into histone and nonhistone fractions by a procedure which completely avoids exposure to extremes of pH. These fractions have been characterised by amino acid analysis and gel electrophoresis. Antisera have been prepared against whole chromatin and against the two chromosomal protein fractions. — A new method is described for the preparation of Drosophila salivary chromosomes. This method employs microdissection techniques and completely avoids the use of acid fixatives. Preservation of fine structure in these preparations is comparable to, if not better than, that in classical acid-fixed preparations. Antisera against embryo chromatin and chromosomal protein fractions react with the salivary chromosome preparations. These reactions exhibit selectivity with different chromosomal structures. Evidence is presented suggesting a specific distribution of protein antigens along the chromosome.

The effect of 20-hydroxyecdysone on synthesis of chromosomal and cytosol proteins in imaginal discs

Abstract Over 600 cytosol and 300 nonhistone chromosomal proteins of mass-isolated imaginal discs of Drosophila melanogaster have been resolved by two-dimensional electrophoresis. More than half of the nonhistone chromosomal proteins fall into families with effectively constant apparent molecular weight but varying isoelectric points. At least six chromosomal proteins differ distinctly in proportions between embryos and imaginal discs. The synthesis of six cytosol proteins is increased, and one decreased with incubation of the discs in vitro with 20-hydroxyecdysone. Two disc acidic chromosomal proteins are specifically synthesized in the presence of 20-hydroxyecdysone. Their isoelectric points and molecular weights are similar to those of the subunits of vertebrate steroid hormone receptor proteins. However, although ecdysteroid receptor activity is associated with purified chromatin, no ecdysteroid-dependent increase in receptor activity is detected during in vitro culture of discs.

Bands, interbands and puffs in native Drosophila polytene chromosomes are recognized by a monoclonal antibody to an epitope in the carboxy-terminal tail of histone H1

A monoclonal antibody was raised against Drosophila melanogaster histone H1. Immunoscreening of proteolytic cleavage fragments of H1 and of a set of all possible overlapping synthetic octapeptides corresponding to the amino acid sequence of H1, revealed that the antibody recognizes an epitope within the sequence 207VTAAKPKA214 near the centre of the carboxy-terminal tail. This antibody gives positive immunofluorescence over the entire length of native D. melanogaster polytene chromosomes isolated from salivary glands by microdissection at physiological pH and ionic strength. Bands, interbands and puffs are all seen to contain H1. The immunofluorescence over puffs, albeit lower than that over bands and interbands, indicates that chromatin decondensation can occur without complete loss of H1 in these structures. The reaction of the antibody with bands suggests that the segment of the C-terminal tail containing the epitope may be exposed in the condensed 30 nm chromatin filament.

The localisation of an Mr 74,000 major chromatin antigen on native salivary chromosomes of Drosophila melanogaster

An antigen making a major contribution to the immune response to Drosophila melanogaster chromatin resides primarily on a nonhistone charge-class family of proteins of Mr 74,000. Immunofluorescence detects this antigen at interbands, puffs and diffuse bands of D. melanogaster salivary chromosomes isolated without exposure to acid fixatives, and on nucleoplasmic ribonucleoprotein droplets. In the electron microscope, gold labelling reveals the binding of monoclonal antibodies specific for the antigen at chromosomal loci generally bearing putative ribonucleoprotein (RNP) particles. However, the locus 3C 11–12 is remarkable in that it bears putative RNP particles but is virtually unlabelled, suggesting protein specificity at different active loci.