Systemic bone loss and induction of coronary vessel disease in a rat model of chronic inflammation.

Clinically, osteopenia or low bone mass has been observed in a variety of chronic inflammatory diseases, and elevated proinflammatory mediators have implicated this process. The purpose of this study was to develop an in vivo model of bone loss induced by chronic systemic inflammation. Time-release pellets designed to deliver one of three doses of LPS: Low (3.3 microg/day), High (33.3 microg/day), or Placebo over 90 days, were implanted subcutaneously in 3-month-old male Sprague-Dawley rats (n = 8/group). Neutrophil counts, indicative of ongoing inflammation, were elevated (P < 0.05) in both LPS groups at 30 days post-implant and remained significantly elevated in the High dose throughout the 90-day study period. At the end of the study, bone loss occurred in the femur as indicated by decreased bone mineral density (BMD) in both LPS-treated groups, but vertebral BMD was reduced in the High dose animals only. Microcomputed tomography revealed that trabecular bone volume (BV/TV) of the proximal tibial metaphysis tended to be reduced in the High dose LPS group. Deleterious effects on trabecular number (TbN) and trabecular separation (TbSp) were observed in both LPS-treated groups, but only the High dose group reached statistical significance. These alterations in trabecular microarchitecture resulted in compromised biomechanical properties. No changes in cortical thickness, porosity, or area of the tibia midshaft were evident at either dose of LPS. Up-regulation of the proinflammatory mediators, cyclooxygenase (COX)-2, interleukin (IL)-1, and tumor necrosis factor (TNF)-alpha was demonstrated in the metaphyseal region where the deleterious effects of LPS were observed. In addition to these alterations in bone, trichrome staining indicated changes in the coronary arterioles, consistent with vascular disease. Utilization of a LPS time-release pellet appears to provide an in vivo model of chronic inflammation-induced bone loss and a potentially novel system to study concurrent development of osteopenia and vascular disease.

Stress induced in heart and other tissues by rat dermal exposure to JP-8 fuel.

Limited information is available regarding the development of systemic organ stress by dermal exposure to JP-8 fuel. In this study, the systemic stress potential of this fuel is evaluated in a rat model subjected to dermal applications of JP-8 for 7 days at 300 microl per day. Tissue histology indicated that JP-8 induces morphological alterations that suggest that tissue stress in the heart is more substantial than stress in the kidney and liver. Immunoblot analysis of tissues revealed increased levels of the inducible heat shock protein 70 (HSP70) in the heart, kidney, and liver after this dermal JP-8 exposure. This exposure also leads to increased levels of heme oxygenase-1 (HO-1/HSP3) in the liver. Additionally during this exposure, a negative regulator of inflammation, IkappaBalpha (inhibitor of NF-kappaB), was increased in the liver, slightly increased in the kidney, and not increased in the heart. Two regions of the rat brain were also examined and HSP70 and IkappaBalpha were increased in the cerebellum but not significantly increased in the cortex. This study indicates dermal JP-8 exposure causes systemic alterations that are associated with cytoprotective activities (e.g., in the liver) as well as potentially toxic mechanisms (heart and kidney).

A rat model of syngeneic bone marrow transplantation during breast cancer therapy.

The purpose of this study was to develop a breast cancer model in rats, in which myeloablative chemotherapy and syngeneic bone marrow transplantation (SBMT) could be evaluated systematically for therapeutic effect. The Wistar-Furth (WF) DMBA-4 breast cancer cell line transplanted into naive WF rats produced rapidly growing tumors that were lethal within 2 months. SBMT was performed following preparation with a regimen (Bu-Cy), consisting of busulfan 16 mg/kg by gastric gavage on days -3 and -2 followed by 250 mg/kg of cyclophosphamide i.p. on day -1. Marrow was prepared from the femurs of donors and infused i.v. into the recipient on day 0. In all, 15 rats treated with Bu-Cy without marrow died, while 22 of 25 transplanted rats survived. In total, 16 rats with measurable tumors showed tumor responses following transplantation, but tumors recurred and survival was minimally prolonged. Of nine rats transplanted before clinical tumors were detected, five became long-term survivors that resisted further tumor challenge. It was concluded that the DMBA-4 breast cancer in WF rats could serve to evaluate SBMT following myeloablative doses of chemotherapy at various tumor loads. At large tumor loads therapy was not curative, but at low tumor burdens cures were possible and resistance to subsequent tumor challenge was demonstrated. The model may be useful for further studies of stem cell infusion in rodent tumor systems.

Mercuric ion inhibition of eukaryotic transcription factor binding to DNA.

Mercury has harmful effects in both rodents and humans. In rodent tissue culture cells exposed to HgCl(2), the metal ions were observed to concentrate in cell nuclei and to associate with chromatin. Thus, transcription factors and other proteins associated with chromatin are possible targets of mercuric ion toxicity. In this study, mercuric ions were found to inhibit the DNA binding activity of the Cys(2)His(2) zinc finger proteins transcription factor IIIA (TFIIIA) and Sp1. These factors are prototypes of the largest eukaryotic protein superfamily. Neither the presence of excess zinc ions nor beta-mercaptoethanol prevented inhibition by mercuric ions. Mercuric ions also inhibited DNA binding by the non-zinc finger protein AP2. Zinc finger-DNA binding was inhibited when both TFIIIA/5S RNA complex and TFIIIA alone were preincubated with concentrations as low as 15 microM mercuric ion. Inhibition occurred in less than 1 min and was not readily reversible. Mercuric ions also inhibited the digestion of DNA by the restriction enzymes BamHI or EcoRI. Inhibition of transcription factors as well as potentially other DNA binding proteins by micromolar concentrations of mercuric ion suggests additional biochemical mechanisms for mercury toxicity in promoting disease via alterations in gene transcription patterns.

Identification of a transcription factor IIIA-interacting protein.

Transcription factor IIIA (TFIIIA) activates 5S ribosomal RNA gene transcription in eukaryotes. The protein from vertebrates has nine contiguous Cys(2)His(2)zinc fingers which function in nucleic acid binding, and a C-terminal region involved in transcription activation. In order to identify protein partners for TFIIIA, yeast two-hybrid screens were performed using the C-terminal region of Xenopus TFIIIA as an attractor and a rat cDNA library as a source of potential partners. A cDNA clone was identified which produced a protein in yeast that interacted with Xenopus TFIIIA but not with yeast TFIIIA. This rat clone was sequenced and the primary structure of the human homolog (termed TFIIIA-intP for TFIIIA-interacting protein) was determined from expressed sequence tags. In vitro interaction of recombinant human TFIIIA-intP with recombinant Xenopus TFIIIA was demonstrated by immuno-precipitation of the complex using anti-TFIIIA-intP antibody. Interaction of rat TFIIIA with rat TFIIIA-intP was indicated by co-chromatography of the two proteins on DEAE-5PW following fractionation of a rat liver extract on cation, anion and gel filtration resins. In a HeLa cell nuclear extract, recombinant TFIIIA-intP was able to stimulate TFIIIA-dependent transcription of the Xenopus 5S ribosomal RNA gene but not TFIIIA-independent transcription of the human adenovirus VA RNA gene.

Lead inhibition of DNA-binding mechanism of Cys(2)His(2) zinc finger proteins.

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 Cys(2)His(2) 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 microM lead ions and completely inhibited by 10 to 20 microM 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 beta-mercaptoethanol. Inhibition kinetics were fast, progressing to completion in approximately 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 Cys(2)His(2) finger protein, but not by the nonfinger protein AP2. Inhibition of Cys(2)His(2) 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.

Expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and p53 tumor suppressor in dysplastic progression and adenocarcinoma in Barrett esophagus.

BACKGROUND: Barrett esophagus predisposes individuals to esophageal carcinoma, which develops from intermediate stages of tissue dysplasia primarily in the vicinity of the gastroesophageal junction. Understanding the cellular and molecular events in the progression of Barrett esophagus to adenocarcinoma may contribute to its early diagnosis and treatment. Mutation and overexpression of the tumor suppressor p53 have previously been observed in Barrett high grade dysplasia and adenocarcinoma. The expression of the cyclin-dependent kinase (CdK) inhibitor p21 can be up-regulated by p53, resulting in the down-regulation of cell division at the G(1)/S-phase transition. The current study examined the correlation between the expression of p21 and p53 by quantifying their levels during the progression of dysplasia and adenocarcinoma in Barrett esophageal tissues. METHODS: Barrett esophageal tissue samples that were negative or indefinite for dysplasia, contained dysplasia, and contained adenocarcinoma were examined by immunohistochemistry. Paraffin embedded sections of lining and glandular epithelia were adsorbed with primary murine antibodies against human p21 or p53 followed by horseradish peroxidase secondary antibody. An immunoreactivity score for each primary antibody and section was obtained by multiplying a staining intensity factor by the percent of positively stained cells. RESULTS: Nuclear p21 expression was detectable immunohistochemically in Barrett esophagus that was negative for dysplasia, but it was significantly elevated (P

Prevention of rat mammary carcinoma utilizing leuprolide as an equivalent to oophorectomy.

A clinical trial is currently under way to examine the effectiveness of leuprolide as a breast cancer chemopreventive agent and contraceptive. This trial, as well as similar proposed studies, is based on the assumption that leuprolide is as effective as surgical castration in preventing the onset of mammary tumors; however, this has not been well documented in the DMBA animal model. We directly compared leuprolide and oophorectomy in this model and examined a combined therapy of leuprolide/bromocriptine. Twenty-seven day old female Sprague-Dawley rats were randomly allocated into one of eight groups. All rats received a 20-mg dose of DMBA at the age of 55 days. Group 1 (n = 10), no treatment; Group 2 (n = 9), leuprolide (100 microg/kg/day) for eight weeks beginning four weeks prior to DMBA; Group 3 (n = 10), oophorectomy four weeks prior to DMBA with replacement estrogen beginning four weeks following DMBA. Estrogen replacement was achieved with a 0.05-mg estradiol tablet releasing 0.833 microg/day over a 60-day period. Group 4 (n = 10), leuprolide (100 microg/kg/day) initiated two weeks prior to DMBA and continuing for two weeks following DMBA; Group 5 (n = 9), oophorectomy two weeks prior to DMBA with 0.05 mg of estradiol in depot form, releasing 0.833 microg/day, beginning four weeks following DMBA and continuing until week 16 of the study; Group 6 (n = 10), leuprolide (100 microg/kg/day) beginning two weeks prior to DMBA and continuing for the duration of the experiment; Group 7 (n = 10), leuprolide (100 microg/kg/day) for eight weeks beginning two weeks prior to DMBA; Group 8 (n = 9), leuprolide (100 microg/kg/day) and bromocriptine (83 microg/day) for eight weeks beginning two weeks prior to DMBA. At nineteen weeks (15 weeks post DMBA), animals were sacrificed and autopsies performed. One hundred percent of untreated animals developed tumors. No animals undergoing oophorectomy four weeks prior to DMBA or receiving leuprolide four weeks prior to and simultaneously with DMBA developed tumors. In animals pretreated two weeks prior to DMBA with leuprolide or oophorectomy, each group had one animal with tumor development. No tumors developed in the animals receiving ongoing injections of leuprolide. However, one tumor developed in those receiving leuprolide for the first eight weeks beginning two weeks prior to DMBA administration. One animal receiving both leuprolide and bromocriptine developed one tumor. We conclude that chemical oophorectomy (with leuprolide) is as effective as surgical oophorectomy in inhibiting DMBA induced carcinogenesis.

Mechanism of action of RNA polymerase II elongation factor Elongin. Maximal stimulation of elongation requires conversion of the early elongation complex to an Elongin-activable form.

We previously identified and purified Elongin by its ability to stimulate the rate of elongation by RNA polymerase II in vitro (Bradsher, J. N., Jackson, K. W., Conaway, R. C., and Conaway, J. W. (1993) J. Biol. Chem. 268, 25587-25593). In this report, we present evidence that stimulation of elongation by Elongin requires that the early RNA polymerase II elongation complex undergoes conversion to an Elongin-activable form. We observe (i) that Elongin does not detectably stimulate the rate of promoter-specific transcription initiation by the fully assembled preinitiation complex and (ii) that early RNA polymerase II elongation intermediates first become susceptible to stimulation by Elongin after synthesizing 8-9-nucleotide-long transcripts. Furthermore, we show that the relative inability of Elongin to stimulate elongation by early elongation intermediates correlates not with the lengths of their associated transcripts but, instead, with the presence of transcription factor IIF (TFIIF) in transcription reactions. By exploiting adenovirus 2 major late promoter derivatives that contain premelted transcriptional start sites and do not require TFIIF, TFIIE, or TFIIH for transcription initiation, we observe (i) that Elongin is capable of strongly stimulating the rate of synthesis of trinucleotide transcripts by a subcomplex of RNA polymerase II, TBP, and TFIIB and (ii) that the ability of Elongin to stimulate synthesis of these short transcripts is substantially reduced by addition of TFIIF to transcription reactions. Here we present these findings, which are consistent with the model that maximal stimulation of elongation by Elongin requires that early elongation intermediates undergo a structural transition that includes loss of TFIIF.

Differential requirements for basic amino acids in transcription factor IIIA-5S gene interaction.

Basic amino acids Arg, Lys, and His in the Cys2His2 zinc fingers of transcription factor IIIA (TFIIIA) potentially have important roles in factor binding to the extended internal control region (ICR) of the 5S ribosomal gene. Conserved and non-conserved basic residues in the N-terminal fingers I, II, III and the more C-terminal fingers V and IX were analyzed by site-directed mutagenesis and DNase I protection in order to assess their individual requirement in the DNA-binding mechanism. In the DNA recognition helix of finger II, the conserved Arg at position 62 (N-terminal side of the first zinc-coordinating histidine) was changed to a Leu or Gln. Both the R62L and R62Q mutations inhibited Xenopus TFIIIA-dependent DNase I footprinting along the entire 5S gene ICR. When His-58 (non-conserved basic residue with DNA-binding potential in the same helical region) was changed to a Gln, the mutated protein was able to protect the ICR from DNase I digestion. Therefore, Arg-62 is individually required for TFIIIA binding over the entire ICR whereas His-58 is not. Fingers V and IX have conserved Arg residues in positions identical to Arg-62 in finger II (Arg-154 in finger V and Arg-271 in finger IX). When these residues were changed to Leu and Ile respectively, TFIIIA-dependent DNase I protection was observed along the entire 5S gene ICR. These results indicate differing DNA-binding mechanisms by the N-terminal fingers versus the C-terminal fingers at the level of individual amino acid-nucleotide interactions. In the N-terminal finger I, the conserved Lys at position 11 outside the recognition helix and a conserved hydrophobic Trp at position 28 within the helix were changed to an Ala and Ser respectively. The K11A change inhibited TFIIIA-dependent DNase I protection to a much greater extent than the W28S change.

Prevention of DMBA-induced rat mammary carcinomas comparing leuprolide, oophorectomy, and tamoxifen.

Leuprolide, a gonadotropin releasing hormone agonist, is currently being evaluated in a pilot study of premenopausal women for the prevention of breast cancer. However, little data is available regarding the efficacy of leuprolide in experimental animal models of carcinoma when administered prior to the carcinogen. In the present study the capacity of leuprolide to prevent tumor development was evaluated by comparing its pretreatment effects in the DMBA-induced rat mammary carcinoma model to pretreatment with tamoxifen and oophorectomy. Fifty-five day old, female Sprague-Dawley rats were randomly allocated to one of four groups: 1) no treatment; 2) oophorectomy two weeks prior to DMBA; 3) leuprolide, 40 microg/kg/day; and 4) tamoxifen, 10 mg/kg/week. All animals received four 5 mg doses of DMBA for a total dose of 20 mg. Leuprolide and tamoxifen treatments began two weeks prior to DMBA and ended one week after DMBA administration. Animals were assessed weekly to determine palpable tumor onset, number, size, and volume. At the conclusion of the study (16 weeks), autopsies were performed and tumor tissue was collected for confirmation of malignancy. Seventy-eight percent of the untreated rats developed tumors. No tumors developed in the oophorectomy group, while the number of rats with tumors was significantly reduced (p<0.05) with both leuprolide (30%) and tamoxifen (21.9%) compared to controls (77.8%). There were no significant differences in the tumor number for each tumor-bearing rat or in tumor volume between treated and control groups. Using our dosage regimen, 'chemical oophorectomy' with leuprolide was not as effective as surgical oophorectomy in the prevention of chemical carcinogenesis by DMBA but was comparable to the results obtained with tamoxifen.

Molecular biology of vertebrate transcription factor IIIA: cloning and characterization of TFIIIA from channel catfish oocytes.

TFIIIA regulates 5S rRNA synthesis and is the prototype of the Cys2His2 superfamily of zinc finger proteins. Because the TFIIIA aa sequence is highly diverged, elucidating species variation in this factor will yield insights into how zinc fingers bind DNA and how this protein regulates RNAPIII transcription. This study reports the identification, cloning and functional divergence of oocyte TFIIIA from the channel catfish. Catfish oocyte TFIIIA was identified by its association with 5S rRNA in immature ovarian tissue, its molecular weight, and by peptide sequence similarities with Xenopus TFIIIA. The cDNA for this factor was cloned by degenerate PCR and found to code for nine Cys2His2 zinc fingers and a C-terminal tail; only about 40% aa sequence identity was observed with Xenopus TFIIIA. The N-terminal region of catfish TFIIIA contains the oocyte-specific initiating Met amino acid and accompanying conserved residues found in amphibian TFIIIAs but not found in yeast or human TFIIIAs. Catfish TFIIIA lacks the conserved transcription activation domain in its C-terminal tail found in amphibian and human TFIIIA. Catfish TFIIIA was able to bind the catfish and Xenopus 5S RNA genes but did not efficiently promote 5S gene transcription in a rodent RNAPIII transcription system, as did Xenopus TFIIIA. Amino acid conservation in catfish, amphibian, and human TFIIIA zinc fingers allows deduction of possible finger recognition helix alignments along the conserved 5S gene ICRs. For the three N-terminal fingers, this leads to deduction of a compact polypeptide structure with conserved basic residues contacting conserved G nts in the 5S gene C box.

Genes for murine Y1 and Y3 Ro RNAs have class 3 RNA polymerase III promoter structures and are unlinked on mouse chromosome 6.

Murine YRNAs, which are components of the conserved Ro ribonucleoprotein (RNP) complex, have been identified by enzymatic RNA sequencing. Mouse Y1 (mY1) and Y3 (mY3; originally named mY2) RNAs share 97 and 95% identity to the human Y1 and Y3 RNAs, respectively. TATA-like sequences, Proximal Sequence Elements, and octamer sequences, which are upstream promoter element motifs indicative of Class 3 RNA Polymerase III (RNAPIII) transcribed genes, are found upstream of both the putative mY1 and mY3 coding regions. Further, these elements are strikingly conserved both in sequence and position relative to known Class 3 genes and to human YRNA genes. Inhibition of transcription in vitro by 200 micrograms/ml but not 1 microgram/ml of alpha-amanitin indicates transcription of the mouse YRNA genes by RNAPIII. Southern blot of C57BL/6J and Mus spretus murine genomic DNA with mY1 and mY3 gene-specific probes suggests that these genes are single copy in the mouse genome. Finally, gene mapping with a (C57BL/6J x SPRET/Ei)F1 x SPRET/Ei mouse interspecific backcross DNA panel localizes the mY1 gene to the distal end of mouse chromosome 6, close to the motheaten (me) autoimmunity locus. The mY3 gene maps to the proximal end of mouse chromosome 6 very close to the T cell receptor beta locus, in a region homologous to human chromosome 7 where the human YRNA genes have been mapped.

Inhibition of transcription factor IIIA-DNA interactions by xenobiotic metal ions.

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.

Comparison of the sequence and structure of transcription factor IIIA from Bufo americanus and Rana pipiens.

Amino acid (aa) sequences of transcription factor IIIA (TFIIIA) from the toad, Bufo americanus, and the grass frog, Rana pipiens, were determined by cDNA cloning and DNA sequencing. The 3'-untranslated regions of the cDNAs reveal that the TFIIIA gene polyadenylation signal is ATTAAA, rather than the conventional AATAAA. The B. americanus and R. pipiens proteins share about 60% aa sequence homology with each other and with Xenopus laevis TFIIIA. Although these results indicate that TFIIIA has more sequence variation than other DNA-binding proteins, a number of conserved features are evident and of likely functional significance. These include potential guanine nucleotide-binding sites at arginines in zinc fingers (ZnF) II, V, and IX, acidic residues between metal-coordinating cysteines, and a basic region in the C-terminal tail possibly involved in transcription promotion. Sequence similarity also exists in an aa stretch bridging the ninth ZnF and C-terminal tail of both TFIIIA and the 5S RNA-binding protein, p43. DNase I protection analyses demonstrate that B. americanus and R. pipiens TFIIIA interact with the internal control region (ICR) of the Xenopus borealis 5S RNA-encoding gene (5S) in different manners: the B. americanus interaction is similar to X. laevis TFIIIA, protecting the entire 5S gene ICR (nt +96 to +43) from DNase I digestion, whereas the R. pipiens TFIIIA strongly protects the ICR from nt +96 up to +78 and less strongly from +78 to +43. Possibly accounting for the binding differences observed, R. pipiens and R. catesbeiana oocyte 5S RNAs (and by inference 5S genes) were found to contain a G or U at nt position 50 while B. americanus, X. laevis, and other eukaryotic 5S RNAs have an A in the analogous position (nt 53 in generalized eukaryotic structure).

Structural elements in the N-terminal half of transcription factor IIIA required for factor binding to the 5S RNA gene internal control region.

Zinc binding domains and the conserved Thr-Gly-Glu-Lys (TGEK) tetrapeptide in the N-terminal half of transcription factor IIIA (TFIIIA) were subjected to in vitro mutagenesis to biochemically assess their role in factor interaction with the 5S gene internal control region (ICR). TFIIIA containing a Leu in place of His33 in the Cys2His2 zinc binding site of finger I lost the ability to protect the entire 5S RNA gene ICR (nucleotides +96 to +43) from DNase I digestion. Thus, mutation of one potential zinc ligand in the N-terminal finger inhibited specific DNA binding by the N-terminal as well as downstream fingers. Cooperativity apparently exists among TFIIIA zinc fingers in metal binding/finger folding and DNA binding. Substituting a Ser for Gly69 or a Glu for Lys 71 in the conserved TGEK tetrapeptide in finger II of TFIIIA resulted in the loss of DNA binding. A Gly-dependent bend structure and a terminal positive charge in this tetrapeptide are important for TFIIIA interaction with DNA. Whereas TFIIIA with a Ser substituted for Cys20 in finger I (proposed zinc ligand) did not protect the ICR from DNase I digestion, TFIIIA containing a Ser substituted for Cys35 (not a proposed zinc ligand) retained the ability to bind the ICR. When Cys112 or Cys 164 (proposed zinc ligands in fingers IV and VI) were replaced by Ser, the DNase I footprint patterns afforded by the respective mutant proteins were similar, protection on the ICR from about nucleotides +96 up to +78. A similar pattern was obtained with a TFIIIA mutant in which fingers V, VI, VII, and a portion of VIII were deleted. Maintenance of zinc coordination spheres in necessary for DNA binding by downstream fingers. The six fingers comprising the N-terminal half of TFIIIA appear to act in two groups of three with binding of the second group dependent upon initial binding of the N-terminal group to the +90 to +80 region of the 5S gene ICR.

Identification of a TFIIIA binding site on the 5' flanking region of the TFIIIA gene.

Xenopus transcription factor IIIA (TFIIIA) regulates 5S ribosomal RNA gene transcription in a positive manner by binding to the internal control region (ICR) of the 5S RNA gene. The present study reports the identification of a TFIIIA binding site on the 5' flanking region of the Xenopus laevis gene that codes for the synthesis of this transcription factor. This TFIIIA binding site (deduced from TFIIIA-dependent alterations in DNase I protection patterns) extends on the 5' flanking region from about nucleotide -326 to -264 on the non-coding strand and from -331 to -271 on the coding strand. The affinity of TFIIIA for the 5' flanking region of its own gene is less than that for the 5S RNA gene but within the same order of magnitude. A sequence similarity between this newly identified binding site and the 5S gene ICR is the presence of purine-rich tracts. EDTA chelation of TFIIIA inhibits binding to this 5' flanking element indicating, as with the 5S RNA gene, zinc is required for DNA binding specificity. When TFIIIA is bound to 5S RNA in the 7S particle, the protein does not bind to this 5' flanking region, an observation similar to that observed with binding to the 5S RNA gene. These results indicate TFIIIA is using similar nucleic acid binding domain(s) for interaction with the 5S gene, 5S RNA, and the 5' flanking region of its own gene. This upstream DNA region to which TFIIIA binds has been previously shown to contain a negative and a positive regulatory element for transcription of the TFIIIA gene (Scotto, K.W., Kaulen, H., and Roeder, R.G., 1989, Genes & Develop. 3, 651-662). The present results indicate TFIIIA binds to the negative control element located at -306 to -289 and possibly interacts/interferes with another transcription factor which binds to the positive control element extending from -271 to -253.

Sequence variation in transcription factor IIIA.

Previous studies characterized macromolecular differences between Xenopus and Rana transcription factor IIIA (TFIIIA) (Gaskins et al., 1989, Nucl. Acids Res. 17, 781-794). In the present study, cDNAs for TFIIIA from Xenopus borealis and Rana catesbeiana (American bullfrog) were cloned and sequenced in order to gain molecular insight into the structure, function, and species variation of TFIIIA and the TFIIIA-type zinc finger. X. borealis and R. catesbeiana TFIIIAs have 339 and 335 amino acids respectively, 5 and 9 fewer than X. laevis TFIIIA. X. borealis TFIIIA exhibited 84% sequence homology (55 amino acid differences) with X. laevis TFIIIA and R. catesbeiana TFIIIA exhibited 63% homology (128 amino acid changes) with X. laevis TFIIIA. This sequence variation is not random; the C-terminal halves of these TFIIIAs contain substantially more non-conservative changes than the N-terminal halves. In particular, the N-terminal region of TFIIIA (that region forming strong DNA contacts) is the most conserved and the C-terminal tail (that region involved in transcription promotion) the most divergent. Hydropathy analyses of these sequences revealed zinc finger periodicity in the N-terminal halves, extreme hydrophilicity in the C-terminal halves, and a different C-terminal tail hydropathy for R. catesbeiana TFIIIA. Although considerable sequence variation exists in these TFIIIA zinc fingers, the Cys/His, Tyr/Phe and Leu residues are strictly conserved between X. laevis and X. borealis. Strict conservation of only the Cys/His motif is observed between X. laevis and R. catesbeiana TFIIIA. Overall, Cys/His zinc fingers in TFIIIA are much less conserved than Cys/Cys fingers in erythroid transcription factor (Eryf 1) and also less conserved than homeo box domains in segmentation genes. The collective evidence indicates that TFIIIA evolved from a common precursor containing up to 12 finger domains which subsequently evolved at different rates.