7S particles containing 5S RNA and the transcription regulatory protein factor A have been purified to near homogeneity from Xenopus laevis oocytes. The binding of the transcription factor to the Xenopus borealis somatic 5S RNA gene has been monitored by quantitating the DNase I protection patterns of the protein-DNA interaction. Under stoichiometric binding conditions--i.e., when all added DNA binds to the factor--two protein molecules are required to saturate the 5S RNA gene. Under equilibrium binding conditions, titration of the 5S RNA gene with factor A results in a sigmoidal binding isotherm suggesting a cooperative interaction; half-saturation of binding is observed at a free factor A concentration of 1 nM. Cooperative binding between factor A and the 5S RNA gene may contribute to the stability of the transcription apparatus and its maintenance during cell division.

Transcription factor IIIA (TFIIIA), a cysteine-rich regulatory protein, is the prototype for the largest known superfamily of eukaryotic transcription factors. Members of the TFIIIA superfamily contain Cys2His2 zinc finger domains responsible for nucleic acid binding. Xenobiotic metal ions, which lack known biological function, were previously used as probes for the structure and function of steroid hormone receptors which contain Cys2Cys2 zinc finger domains. Structural alterations in cysteine-rich regulatory proteins by such ions in vivo might potentiate carcinogenesis and other disease processes. In the present study cadmium and other xenobiotic metal ions were used to probe the structure and function of TFIIIA. The specific interaction of TFIIIA with the internal control region (ICR) of the 5S RNA gene, as assayed by DNase I protection, was inhibited by Cd2+ ion concentrations of > or = 0.1 microM. Aluminum ions were also found to inhibit the TFIIIA-5S RNA gene interaction, albeit at higher concentrations (> or = 5 microM). Inhibition by either metal ion was not readily reversible. Other xenobiotic metal ions, such as mercury or cesium, were not found to be inhibitory under these conditions. None of these ions at the concentrations used in this study affected the ability of DNase I to digest DNA or restriction enzymes to specifically cleave DNA. Preincubation of TFIIIA bound to 5S RNA with either Cd2+ or Al3+ resulted in subsequent DNA binding upon dilution and RNA removal, whereas preincubation of free TFIIIA with the metal ions resulted in inhibition of subsequent DNA binding. Because 5S rRNA also binds the TFIIIA zinc finger domains, these results indicate that the 5S RNA bound to TFIIIA protects the protein from metal inhibition and implicates the zinc fingers in the inhibition mechanism. The nature of the footprint inhibition indicates that the N-terminal fingers of TFIIIA are affected by the metal ions. Cd2+ and Al3+ ions also inhibited the ability of TFIIIA to bind complementary single-stranded DNA and promote renaturation, as measured by Tris-phosphate agarose gel electrophoresis. This gel assay is sensitive to DNA conformation and Al3+ ions were found to alter the conformation of single- and double-stranded DNA in this assay. The inhibition of TFIIIA function in vitro by xenobiotic metals offers new insights into the structure and function of TFIIIA and TFIIIA-type zinc finger proteins. Inhibition by Cd2+ occurs at much lower concentrations than previously observed with steroid hormone receptors and suggests that Cys2His2 zinc finger proteins may be especially sensitive to such agents in vivo.

The association of lead with chromatin in cells suggests that deleterious metal effects may in part be mediated through alterations in gene function. To elucidate if and how lead may alter DNA binding of cysteine-rich zinc finger proteins, lead ions were analyzed for their ability to alter the DNA binding mechanism of the Cys₂His₂ zinc finger protein transcription factor IIIA (TFIIIA). As assayed by DNase I protection, the interaction of TFIIIA with the 50-bp internal control region of the 5S ribosomal gene was partially inhibited by 5 μM lead ions and completely inhibited by 10 to 20 μM lead ions. Preincubation of free TFIIIA with lead resulted in DNA-binding inhibition, whereas preincubation of a TFIIIA/5S RNA complex with lead did not result in DNA-binding inhibition. Because 5S RNA binds TFIIIA zinc fingers, this result is consistent with an inhibition mechanism via lead binding to zinc fingers. The complete loss of DNase I protection on the 5S gene indicates the mechanism of inhibition minimally involves the N-terminal fingers of TFIIIA. Inhibition was not readily reversible and occurred in the presence of an excess of β-mercaptoethanol. Inhibition kinetics were fast, progressing to completion in ∼5 min. Millimolar concentrations of sulfhydryl-specific arsenic ions were not inhibitory for TFIIIA binding. Micromolar concentrations of lead inhibited DNA binding by Sp1, another Cys₂His₂ finger protein, but not by the nonfinger protein AP2. Inhibition of Cys₂His₂ zinc finger transcription factors by lead ions at concentrations near those known to have deleterious physiological effects points to new molecular mechanisms for lead toxicity in promoting disease.

Footprint competition assays are utilized to study the binding of Xenopus transcription factor A to a variety of single-stranded nucleic acids. The addition of Xenopus oocyte, yeast, or wheat germ 5S RNA as footprint competitors reveals that factor A binds these 5S RNAs with similar affinity. In contrast, factor A does not bind to E.coli 5S RNA or wheat germ tRNA in this assay. Factor A binding to single stranded DNA is also examined using footprint competition. Factor A binds preferentially to non-specific single stranded (M13) DNA versus double stranded (pBR322) DNA. Factor A binds equally well to single stranded DNA fragments containing either the coding or non-coding strands of the 5S RNA gene. Using single stranded M13 DNA as a competitor, the factor A-5S RNA gene complex is found to dissociate with a half-life of 5-6 min.

Objective: The aim of this study is to identify biomarkers in sera of pancreatic cancer patients using mass spectrometry (MS) approaches. Methods: Sera from patients diagnosed with pancreatic adenocarcinoma and sera from normal volunteers were subjected to gel electrophoresis to resolve and quantify differences in protein levels. Protein bands that differed quantitatively were digested with trypsin, and peptides were identified by electrospray ionization (ESI) ıon-trap tandem MS. Mass spectra were also collected directly from pancreatic cancer sera as well as healthy control sera using ESI-MS. Results: Three large-mass proteins were found to be elevated in pancreatic cancer sera versus normal sera, α-2 macroglobulin, ceruloplasmin, and complement 3C. Complement 3C is a major regulator of inflammatory responses. The ESI-MS of human pancreatic cancer sera versus normal sera revealed greater heterogeneity in cancer sera than control sera, especially in the low-mass region. Bootstrapping statistical analysis identified 20 low-mass serum peaks that correlated with control sera and 20 different peaks that correlated with pancreatic cancer sera. Conclusions: The fact that inflammation-sensitive proteins were identified as increased in pancreatic cancer sera supports the hypothesis that inflammatory-driven processes are involved in pancreatic carcinogenesis. Liquid ESI-MS analyses of sera hold promise for future pancreatic cancer blood tests as well as for understanding mechanisms of pancreatic carcinogenesis. The variability observed between the low-mass regions of normal versus pancreatic cancer spectra may aid in diagnosis and therapy.