Mapping sequence specific DNA-protein interactions: a versatile, quantitative method and its application to transcription factor XF1.

We have developed a method for the quantitative, exhaustive sequence specificity determination of DNA-binding proteins. The QuESSD method overcomes the limitations inherent in other published in vitro selection methods, not only defining the consensus sequence, but also quantifying the effect on DNA-protein affinity of replacing each base in the recognition domain with every other base. The features distinguishing this method from other in vitro selection approaches are: (1) instead of synthesizing one target oligonucleotide population containing a long randomized domain, we synthesize several oligonucleotide populations, each randomized at two positions. (2) Instead of carrying out several cycles of selection and amplification, we carry out a single cycle. (3) We have developed data collection and analysis procedures that eliminate artifacts and allow generation of quantitative results. The QuESSD method yields accurate measures of: (a) the selectivity of the protein for each base at each position within the recognition domain (normalized relative selectivity), (b) the contributions of individual sites within the recognition domain to the binding affinity (selectivity variance), (c) the relative binding affinity of any given sequence (global selectivity). We confirmed results by (1) tabulating directly the frequency of appearance of individual species in the pool of protein-bound oligonucleotides by cloning and sequencing individual oligonucleotides, and (2) competition EMSA analysis of oligonucleotides designed on the basis of QuESSD data. We have used this method to map the sequence specificity of the nuclear protein XF1 and to distinguish the sequence specificities of XF1 and the AH receptor complex, both of which bind to XRE1, a xenobiotic responsive element (XRE) located upstream of the CYP1A1 gene. Using data obtained by the QuESSD method, we designed oligonucleotides specific for XF1 or for the AH receptor, and prepared CAT reporter gene constructs carrying these oligonucleotides, or wild-type XRE1, upstream of a minimal promoter. Transfection studies using these constructs indicated that XF1 can function as a weak activator of basal transcription, and can, under some circumstances, compete with the AH receptor for binding to XRE1.

Detection of antimicrofilarial ES antigen-antibody in immune complexes in Bancroftian filariasis by enzyme immunoassay.

An enzyme immunoassay using penicillinase conjugated to Wuchereria bancrofti microfilarial ES antigen has been developed to detect specific antibody in circulating immune complexes in Bancroftian filariasis. Immune complexes were prepared by 3% polyethylene glycol (PEG) precipitation. 44 sera belonging to different groups were tested. 16 of 19 clinical filarial and two of 16 endemic normal sera but none of the non-endemic normal sera showed the presence of antimicrofilarial ES antigen-antibody in immune complexes.

Analysis of menstrual records of women immunized with anti-hCG vaccines inducing antibodies partially cross-reactive with hLH.

Menstrual data of 13 control subjects and 88 subjects immunized with three beta-hCG-based vaccine formulations were analysed. Immunization did not change the menstrual regularity; bleeding days were normal (3-7 days) and 89% of the menstrual cycles were within the normal range of 22-35 days. Irregular (short or long) cycles were observed in both immunized and control groups. These were, however, unrelated to prevailing anti-hCG antibody titres or to cross-reactivity of antibodies with hLH.

Suppression of Con A induced lymphocyte transformation by sera from filarial patients with clinical manifestations.

Reduced lymphocyte transformation to Wuchereria bancrofti microfilariae excretory-secretory antigen and Con A were observed in clinical filarial patients. Pre-incubation of normal human peripheral blood mononuclear cells with sera from filarial patients with clinical manifestations such as hydrocele and elephantiasis suppressed Con A induced responses. Effect of fractionated clinical filarial serum on Con A induced lymphocyte transformation showed that the inhibitory activity was associated with high molecular weight serum fraction.

Antigenic analysis of excretory-secretory products of Wuchereria bancrofti and Brugia malayi infective larval forms by SDS-PAGE.

Excretory-secretory (ES) products of W. bancrofti and the closely related B. malayi infective larval forms were analysed for their antigenic activity by SDS-PAGE followed by Western blotting as well as by gel elution-sandwich ELISA using filarial serum immunoglobulin-G (FSIgG) as a capture antibody. In W. bancrofti infective larval ES products, the protein molecules of 66, 46, 35, 33, 30 and 14 kDa molecular wt. showed antigenic activity by immuno blotting technique. In sandwich ELISA technique eventhough all SDS-PAGE fractions except ESA 6 (55-47 kDa) showed antigenic positivity, the fractions ESA 8 (37-31 kDa) and ESA 9 (31-25 kDa) showed high reciprocal antigen titre of 262144 and 32768 respectively. In B. malayi infective larval ES products, the protein molecules of 109, 102, 97 and 77 kDa molecular wt. showed reactivity with FSIgG by blotting technique, where as in sandwich ELISA except ESA 7 (47-37kDa), all fractions showed antigenic positivity. However, these fractions failed to show high antigen titre similar to W. bancrofti ES products with FSIgG.

Antiestrogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin are mediated by direct transcriptional interference with the liganded estrogen receptor. Cross-talk between aryl hydrocarbon- and estrogen-mediated signaling.

Aryl hydrocarbon receptor (AhR) ligands have diverse biological effects including striking antiestrogenic activity. We have investigated at the molecular level the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We show that the previously documented TCDD-mediated decrease in estradiol-inducible gene products such as cathepsin D (cat D) is due to a sharp decline in mRNA accumulation despite any change in estrogen receptor (ER) mRNA levels. The decline in cat D mRNA level is most likely due to a decrease in transcription of the cat D gene since TCDD blocks the ability of ER to transactivate from an estrogen response element. AhR is required for this activity as TCDD is no longer antiestrogenic in a mutant cell line that is deficient in functional AhR. We provide evidence that the loss of transactivation potential by ER in the presence of TCDD is due to a sharp decrease in its ability to bind to an estrogen response element. Reciprocally, estradiol treatment blocked TCDD-induced accumulation of CYP1A1 mRNA and AhR-mediated activation of the CYP1A1 promoter. This is due to the ability of liganded ER to interfere with the binding of AhR to the xenobiotic response element. These results provide a molecular mechanism for the antiestrogenic effects of TCDD and demonstrate the presence of a two-way crosstalk between the intracellular signaling pathways involving estrogens and aryl hydrocarbons.

The utility of human filarial serum in the detection of circulating antigen.

The utility of human filarial serum immunoglobulin (FSI) in detecting circulating antigen in filarial sera was studied by counter immunoelectrophoresis (CIEP) and the indirect haemagglutination test (IHAT). CIEP was found to be better than IHAT. 23 out of 30 sera from persons with microfilaeremia and one of 30 clinical cases of filariasis, but none of the normal sera or sera from those with helminths other than filariae, showed the presence of circulating filarial antigen in CIEP. FSI was fractionated by DEAE- Sephadex A-50 column chromatography and the antibody active in CIEP was found to be IgG in nature.