Characterization of a bovine intestinal myofibroblast cell line and stimulation using phytoglycogen-based nanoparticles bound to inosine monophosphate.

The goal of the present study was to characterize a novel bovine intestinal myofibroblast (BT-IMF) cell line isolated from a fetal bovine intestine. This cell type is of importance as intestinal myofibroblasts play a key role in controlling intestinal epithelial cell proliferation, intestinal regulation, wound healing, epithelial cell turnover, and structural support. The present work demonstrates that BT-IMF cells could be successfully cryopreserved and thawed and cultured past 25 passages. Immunocytochemical staining of the BT-IMF cell line was positive for vimentin and smooth muscle actin (α-SMA) and negative for pancytokeratin, suggesting that the cells are myofibroblastic in type. Growth kinetic experiments demonstrate that hydrocortisone negatively impacts BT-IMF growth and non-essential amino acids enhance its proliferation. Inosine monophosphate (IMP) is a dietary nucleotide and is essential for supporting animal health. Stimulation with IMP bound to a novel phytoglycogen-based nanocarrier (IMP-NP) showed enhanced cell proliferation. BT-IMF provides a new tool for studying bovine cells in vitro and may be of particular interest for cultured meat manufacturing in the future.

Psychological outcomes of evening and night closed-loop insulin delivery under free living conditions in people with Type 1 diabetes: a 2-month randomized crossover trial.

AIM: To assess the impact on fear of hypoglycaemia and treatment satisfaction with an artificial pancreas system used for 2 consecutive months, as well as participant acceptance of the artificial pancreas system. METHODS: In a randomized crossover trial patient-related outcomes associated with an evening-and-night artificial pancreas and sensor-augmented pump therapy were compared. Both intervention periods lasted 8 weeks. The artificial pancreas acceptance questionnaire (range 0-90, higher scores better), Hypoglycaemia Fear Survey II (range 0-72, higher scores worse) and Diabetes Treatment Satisfaction Questionnaire (range 0-36, higher scores better) were completed by 32 participants. Semi-structured interviews were conducted after study completion in a subset of six participants. Outcomes were compared using a repeated-measures anova model or paired t-test when appropriate. RESULTS: The total artificial pancreas acceptance questionnaire score at the end of the artificial pancreas period was 69.1 (sd 14.7; 95% CI 63.5, 74.7), indicating a positive attitude towards the artificial pancreas. No significant differences were found among the scores at baseline, end of sensor-augmented pump therapy period or end of the artificial pancreas period with regard to fear of hypoglycaemia [28.2 (sd 17.5), 23.5 (sd 16.6) and 23.5 (sd 16.7), respectively; P = 0.099] or diabetes treatment satisfaction [29.0 (sd 3.9), 28.2 (sd 5.2) and 28.0 (sd 7.1), respectively; P = 0.43]. Themes frequently mentioned in the interviews were 'positive effects at work', 'improved blood glucose', 'fewer worries about blood glucose', but also 'frequent alarms', 'technological issues' and 'demand for an all-in-one device'. CONCLUSIONS: The psychological outcomes of artificial pancreas and sensor-augmented pump therapy were similar. Current artificial pancreas technology is promising but user concerns should be taken into account to ensure utility of these systems.

Effects of environmental factors on microbial induced calcium carbonate precipitation.

AIMS: To gain an understanding of the environmental factors that affect the growth of the bacterium Sporosarcina pasteurii, the metabolism of the bacterium and the calcium carbonate precipitation induced by this bacterium to optimally implement the biological treatment process, microbial induced calcium carbonate precipitation (MICP), in situ. METHODS AND RESULTS: Soil column and batch tests were used to assess the effect of likely subsurface environmental factors on the MICP treatment process. Microbial growth and mineral precipitation were evaluated in freshwater and seawater. Environmental conditions that may influence the ureolytic activity of the bacteria, such as ammonium concentration and oxygen availability, as well as the ureolytic activities of viable and lysed cells were assessed. Treatment formulation and injection rate, as well as soil particle characteristics are other factors that were evaluated for impact on uniform induction of cementation within the soils. CONCLUSIONS: The results of the study presented herein indicate that the biological treatment process is equally robust over a wide range of soil types, concentrations of ammonium chloride and salinities ranging from distilled water to full seawater; on the time scale of an hour, it is not diminished by the absence of oxygen or lysis of cells containing the urease enzyme. SIGNIFICANCE AND IMPACT OF STUDY: This study advances the biological treatment process MICP towards field implementation by addressing key environmental hurdles faced with during the upscaling process.

[Sustained weight loss 2 years after laparoscopic adjustable gastric banding for morbid obesity]. / Duurzaam gewichtsverlies 2 jaar na laparoscopische maagbandplaatsing wegens morbide obesitas.

OBJECTIVE: To analyse the results of the laparoscopic adjustable gastric banding (LAGB) procedure for morbid obesity. DESIGN. Retrospective, descriptive. METHOD: From November 1, 1995 to May 31, 2005, laparoscopic adjustable banding surgery was performed in St. Antonius Hospital, Nieuwegein, the Netherlands, in 411 patients. Inclusion criteria were BMI > or = 40 kg/ m(2) or BMI > 35 kg/m(2) and severe comorbidity with > 3 attempts at weight loss in the past. Selection, inclusion and follow-up were performed in a specialised, multidisciplinary setting. Height, weight, and complications were prospectively recorded. In 1995-2000 the perigastric surgical procedure was used and in 2000-2005 the pars-flaccida method. RESULTS: The study group consisted of 350 (85%) women and 61 (I5%) men with a median age of 38 years (range 17-60). Out of these 411 patients, the median weight was 133.4 kg, the median overweight, 69.6 kg and the median BMI 46.3 kg/m2. Two years after surgery, data was known for 267 patients where 206 (77%) had a weight loss > 30%, and 7 patients (3%) a weight gain. The median BMI difference was then -10.2 kg/m2 (range +4.7--26.4). The median loss of overweight was 46.3% (+10.00--97.8). The weight loss remained stable in the following years. The most commonly seen complications were fundus slippage (13%) and port-a-cath related complications (7%). These occurred more often in patients who had had the perigastric method surgery than in the parsflaccida surgical method. CONCLUSION: Three quarters of the patients with morbid obesity who received laparoscopic gastric banding surgery had achieved and sustained weight loss at 2 years following surgery. The pars-flaccida method resulted in fewer complications than the perigastric surgical method.

Entrapment in small, enclosed spaces: a case report and points to consider regarding the mechanism of death.

The mechanism of death due to confinement in an enclosed space is usually ascribed to asphyxia from oxygen deprivation. We report the case of the decomposed remains of a 23-year-old man discovered in an unused industrial size refrigerator in which the mechanism of death is heatstroke. The investigation of the death indicates the subject most likely voluntarily entered the refrigerator and for unknown reasons, closed the door. Injuries identified at autopsy and damage to the inside of the structure indicate he struggled to exit the cabinet. The autopsy shows no significant natural disease processes and toxicology studies were negative. The diagnosis of heat stroke typically rests on the evaluation of multiple features, including the age and size of the decedent, the ambient temperature, the medical history of the decedent, whole body hydration, body fat content, alcohol and drug use, medication history, general physical condition, and many other factors. The diagnosis of heatstroke due to confinement in an enclosed container requires evaluation of the heat stress of the container, the heat strain experienced by the individual, autopsy findings suggesting signs of a struggle to exit the container, and other factors. In all such cases, a careful death investigation with correlation of autopsy findings is required to accurately determine the mechanism and cause of death. We suggest that for all such deaths, physiological and environmental factors promoting hyperthermia and heatstroke be considered as a possible mechanism of death, along with those associated with the more obvious danger of asphyxiation.

TFIIAalpha/beta-like factor is encoded by a germ cell-specific gene whose expression is up-regulated with other general transcription factors during spermatogenesis in the mouse.

TFIIAalpha/beta-like factor (ALF) is a testis-specific counterpart of the large subunit of human general transcription factor TFIIA. Northern analysis shows that ALF mRNA first appears in mouse testis at Postnatal Day 14. Similarly, expression of the general transcription factors TBP, TRF2, TFIIAalpha/beta, TFIIAgamma, and TFIIIB(90) is also increased beginning at Postnatal Day 14, suggesting that there is a coordinated induction of many general transcription factors during male germ cell differentiation. Analysis of male germ cells separated by Staput sedimentation shows that ALF is present in pachytene spermatocytes and haploid spermatids. In addition, in situ hybridization experiments with adult mouse testis shows that ALF is present in haploid spermatids. Searches of the human genome sequence database using the basic local alignment search tool reveal that the ALF and TFIIAalpha/beta(GTF2A1) genes are both composed of nine exons, whereas the TFIIAgamma (GTF2A2) gene is composed of five exons. Furthermore, nucleotide and amino acid comparisons among human and mouse ALF, TFIIAalpha/beta, and TFIIAgamma cDNA sequences show that ALF has diverged more rapidly than either TFIIAalpha/beta or TFIIAgamma. Finally, the ALF and SBLF (Stoned B-Like Factor) sequences present in the chimeric SALF cDNA are both present on human chromosome 2, and an analysis of the corresponding genes suggests a model for the formation of SALF.

The role and limitations of the Cairo International Conference on Population and Development.

Population questions have always aroused controversy, but the International Conference on Population and Development (ICPD) which took place in Cairo in September 1994 was particularly contentious. Yet a consensus emerged among stakeholders previously holding quite divergent positions. A "new paradigm" in population policy emerged from the conference which shifted emphasis from a macro concern with rapid population growth to individual rights in sexuality and reproduction. This consensus has been widely praised, but was far from predictable. It was arrived at through a complicated inter-weaving of interests, movements and intellectual trends, as well as owing much to the particular nature of politics--both global and national--at the time. This paper is an analysis of the policy and substantive significance of the ICPD within the context of the history of UN-sponsored population conferences. It explores how the outcome of the conference was perceived by the various interest groups which played a major role in determining its policy directions, and enumerates some of the critiques of its Programme of Action from different perspectives. It reports on progress and obstacles to implementation of its recommendations within a changed political and economic context than that prevailing in 1994.

Functional interactions between an atypical NF-kappaB site from the rat CYP2B1 promoter and the transcriptional repressor RBP-Jkappa/CBF1.

The phenobarbital-inducible rat cytochrome P450 (CYP) 2B1 and 2B2 proteins are encoded by homologous genes whose promoters contain a mammalian-apparent long terminal repeat retrotransposon (MaLR). An NF-kappaB-like site within the MaLR forms multiple protein-DNA complexes with rat liver and HeLa cell nuclear extracts. Using antibody supershift assays, we have identified these complexes as NF-kappaB and RPB-Jkappa/CBF1. Competition assays using a series of single site mutant oligonucleotides reveal that the recognition sites for these two factors overlap. We also show that the CYP2B1/2 NF-kappaB element, but not the Igkappa NF-kappaB element, can repress transcription in vitro when positioned upstream of the heterologous adenovirus major late core promoter. In addition, RBP-Jkappa over-expressed in COS-7 cells repressed expression in vivo from an SV40-luciferase reporter construct that contained the CYP2B1/2 NF-kappaB element. Finally, we observe similar levels of NF-kappaB and RBP-Jkappa binding activities in nuclear extracts prepared from control and phenobarbital-induced rat livers. The results suggest that RBP-Jkappa/CBF1 binds an atypical NF-kappaB site in the CYP2B1/2 promoters and may help to maintain a low level of expression in the absence of inducer.

Level of agreement between opinions of medical examiner investigators and forensic pathologist medical examiners regarding the manner of death.

To evaluate the level of agreement between medical examiner investigators' opinion of the manner of death and what the manner of death was as certified by forensic pathologist medical examiners (MEs), we reviewed the case records stored in a database of all deaths reported to the office of the medical examiner in Fulton County, Georgia. Of 15,771 deaths reported to the office during a 10-year period, a difference exists in 1908 cases. In 900 natural deaths, the investigators recorded 135 accident, 10 homicide, 10 suicide, and 745 undetermined manners of death. In 755 accidental deaths, the investigators recorded 16 natural, 8 homicide, 13 suicide, and 718 undetermined manners of death. In 107 homicides, the investigators recorded 12 natural, 8 accident, 0 suicide, and 87 undetermined manners of death. In 70 suicides, the investigators recorded 9 natural, 9 accident, 3 homicide, and 49 undetermined manners of death. In 61 deaths classified as undetermined, the investigators recorded 25 natural, 13 accident, 17 homicide, and 6 suicide manners of death. In 15 deaths, the discrepancy exists due to an apparent error in the database information. This study confirms a high concordance between investigator and ME opinion regarding manner of death but also documents the need for case review and autopsies by forensic pathologists to confirm the investigators' opinion of the manner of death, determine the manner of death when the investigator selects undetermined, and on occasion, refute the investigators' opinion regarding the manner of death.

Microsomal GST-I: genomic organization, expression, and alternative splicing of the human gene.

In this paper we report the genomic organization of the human microsomal GST-I gene. This gene spans 18 kb, and contains seven exons. Sequences that encode the 155 amino acid open reading frame are present in Exons II, III, IV, the 5'-untranslated region is present in Exons Ia, Ib, Ic, Id, and II, and the 3'-untranslated region is present in Exon IV. Exons Ia, Ib, Ic, Id, and III are alternatively spliced to generate at least six different mGST-I transcripts. The results of EST and PCR analysis show that most mGST-I transcripts terminate within Exon Ib, and primer extension analysis shows these transcripts initiate at three major sites located at 79, 81, and 88 nucleotides upstream of the ATG initiation codon. Sequences surrounding the putative initiation sites are G-C rich, and several Sp1 consensus binding sites were identified. Northern analysis shows that the human GST-I gene is preferentially expressed as a 1.0 kb transcript in liver, and in several other tissues. Finally, a comparison of the mGST-I and PIG12 sequences with those of FLAP, LTC4 synthase, mGST-II, and mGST-III suggests that these proteins are the related products of a dispersed microsomal GST gene superfamily.

Identification of a general transcription factor TFIIAalpha/beta homolog selectively expressed in testis.

In this paper we describe the isolation of a cDNA that encodes a human TFIIAalpha/beta-like factor (ALF). The open reading frame of ALF predicts a protein of 478 amino acids that contains characteristic N- and C-terminal conserved domains separated by an internal nonconserved domain. In addition, a rare ALF-containing cDNA, which possesses an extended N terminus (Stoned B/TFIIAalpha/beta-like factor; SALF) has also been identified. The results of Northern and dot blot analyses show that ALF is expressed almost exclusively in testis; in contrast, TFIIAalpha/beta and TFIIAgamma are enriched in testis but are also widely expressed in other human tissues. Recombinant ALF (69 kDa) and TFIIAgamma (12 kDa) polypeptides produced in Escherichia coli form an ALF/gamma complex that can stabilize TBP-TATA interactions in an electrophoretic mobility shift assay. The ALF/gamma complex is also able to restore transcription from the adenovirus major late promoter using HeLa cell nuclear extracts that have been depleted of TFIIA. Overall, the data show that ALF is a functional homolog of human general transcription factor TFIIAalpha/beta that may be uniquely important to testis biology.

CCR5 coreceptor usage of non-syncytium-inducing primary HIV-1 is independent of phylogenetically distinct global HIV-1 isolates: delineation of consensus motif in the V3 domain that predicts CCR-5 usage.

The cellular tropism of human immunodeficiency virus type 1 (HIV-1) is dependent on utilization of specific chemokine co-receptor: macrophage-tropic/non-syncytium-inducing (NSI) viruses use CCR5, whereas T-cell tropic/syncytium-inducing (SI) viruses preferentially use CXCR4. We have analyzed co-receptor usage of 24 phylogenetically distinct primary HIV-1 isolates representing group M (clades A-F) and group O with known SI and NSI phenotype, using lymphocytes from donor with nonfunctional CCR5 (CCR5-/-; homozygous 32-bp deletion). While all SI isolates infected CCR5-/- lymphocytes (and hence do not require CCR5 for viral entry), all NSI isolates, regardless of clade, did not infect CCR5-/- lymphocytes. Thus, CCR5 expression is required for infection with NSI isolates and the CCR5 usage is independent of viral genotype. To localize the viral determinant involved in CCR5 binding, the V3 sequences across the clades were aligned based on the CCR5 usage. There were conserved uncharged residues at position 11 of V3 (mostly serine/glycine) and negatively charged residues at residue 25 (mostly glutamic/aspartic acid) among all isolates that used CCR5, whereas substitution with arginine or glutamine at these two positions led to usage of a co-receptor other than CCR5. This analysis led us to identify a consensus motif S/GXXXGPGXXXXXXXE/D within the V3 loop that predicts CCR5 co-receptor usage. Most isolates, with exception of one isolate, containing the conserved motif and predicted to utilize CCR5 indeed had an absolute requirement of CCR5 expression for infectibility. Site-directed mutagenesis in the infectious molecular clone further confirmed these results. Taken together, these data provide evidence that sequences within the V3 loop provide important residues that might be directly or indirectly involved in binding to a CCR5 co-receptor.

Interaction of the human T-cell lymphotropic virus type 1 tax transactivator with transcription factor IIA.

The Tax protein of human T-cell lymphotropic virus type 1 (HTLV-1) is a 40-kDa transcriptional activator which is critical for HTLV-1 gene regulation and virus-induced cellular transformation. Tax is localized to the DNA through its interaction with the site-specific activators cyclic AMP-responsive element-binding protein, NF-kappaB, and serum response factor. It has been suggested that the recruitment of Tax to the DNA positions Tax for interaction with the basal transcriptional machinery. On the basis of several independent assays, we now report a physical and functional interaction between Tax and the transcription factor, TFIIA. First, Tax was found to interact with the 35-kDa (alpha) subunit of TFIIA in the yeast two-hybrid interaction system. Importantly, two previously characterized mutants with point mutations in Tax, M32 (Y196A, K197S) and M41 (H287A, P288S), which were shown to be defective in Tax-activated transcription were unable to interact with TFIIA in this assay. Second, a glutathione-S-transferase (GST) affinity-binding assay showed that the interaction of holo-TFIIA with GST-Tax was 20-fold higher than that observed with either the GST-Tax M32 activation mutant or the GST control. Third, a coimmunoprecipitation assay showed that in HTLV-1-infected human T lymphocytes, Tax and TFIIA were associated. Finally, TFIIA facilitates Tax transactivation in vitro and in vivo. In vitro transcription studies showed reduced levels of Tax-activated transcription in cell extracts depleted of TFIIA. In addition, transfection of human T lymphocytes with TFIIA expression vectors enhanced Tax-activated transcription of an HTLV-1 long terminal repeat-chloramphenicol acetyltransferase reporter construct. Our study suggests that the interaction of Tax with the transcription factor TFIIA may play a role in Tax-mediated transcriptional activation.

Fatal stercoral ulcer perforation: case report.

Stercoral ulcers result from severe, prolonged constipation. Stercoral ulcer perforation is a rare event resulting in severe peritonitis and has a very high associated mortality rate. We describe a 78-year-old man with severe constipation associated with stercoral ulcer perforation undiagnosed prior to autopsy.

Heterologous expression of rat liver microsomal glutathione transferase in simian COS cells and Escherichia coli.

The cDNA coding for rat liver microsomal glutathione transferase was subcloned into the mammalian expression vector pCMV-5 and the construct was transfected into, and transiently expressed in, simian COS cells. This resulted in high expression (0.7% of the microsomal protein). The activity towards 1-chloro-2,4-dinitrobenzene in microsomes was 15-30 nmol/min per mg, which increased upon N-ethylmaleimide treatment to 60-200 nmol/min per mg. Control and antisense-vector-treated cells displayed very low activity (3-6 nmol/min per mg). A DNA fragment coding for rat microsomal glutathione transferase was generated by PCR, cloned into the bacterial expression vector pSP19T7LT and transformed into Escherichia coli strain BL21 (DE3) (which contained the plasmid pLys SL). Isopropyl beta-D-thiogalactopyranoside (IPTG; 1 mM) induced the expression of significant amounts of enzymically active protein (4 mg/l of culture as measured by Western blots). The recombinant protein was purified and characterized and found to be indistinguishable from the rat liver enzyme with regard to enzymic activity, molecular mass and N-terminal amino acid sequence. Human liver cDNA was used to obtain the coding region of human microsomal glutathione transferase by PCR. This PCR product was cloned into pSP19T7LT, which, upon induction with IPTG, yielded significant amounts (9 mg/l of culture) of active enzyme in BL21 (DE3) cells. Thus, for the first time, it is now possible to express both human and rat microsomal glutathione transferase in an enzymically active form in Escherichia coli.

Evaluation of a liquid chromatographic method for the determination of fumonisins in corn, poultry feed, and Fusarium culture material.

The performance of a liquid chromatographic method for determining fumonisins in corn, animal feeds, and culture material was evaluated. Efficiencies of extractions with the following solvent systems were determined: acetonitrile-water (50 + 50, v/v), methanol-water (75 + 25, v/v), and 100% water. The acetonitrile solvent gave both higher extraction efficiencies and faster extraction times than the other 2 solvents. Extraction was followed by C18 solid-phase extraction column cleanup. Fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3) were measured by precolumn derivatization with o-phthalaldehyde followed by isocratic separation on a C18 reversed-phase column with a mobile phase of 50 mM potassium dihydrogen phosphate (pH 3.3)-acetonitrile (60 + 40). Commercially prepared poultry feed, corn, and Fusarium spp. corn cultures were analyzed at the following levels: FB1, 1.5 to 15,000 micrograms/g; FB2, 0.5 to 4000 micrograms/g; FB3, and 0.17 to 1,500 micrograms/g. Recoveries were 91-94%, 90-100%, and 81-93% for FB1, FB2, and FB3, respectively. Precision (coefficient of variation) was determined with pooled field samples and ranged from 2% at 19 micrograms/g for FB1 to 9% at 0.17 microgram/g for FB3. Time and pH studies of the formation of the fluorescent derivative and its stability were conducted. Complete reaction occurred at pHs above 7.9, with optimal pH for chromatography between 8.0 and 8.5. No statistically significant response differences were detected for reaction times ranging from 4 to 40 min; however, the detector signal was significantly reduced when reaction times were shorter than 4 min. Chromatograms of samples were free of interferences for all feeds, corn, and culture material tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Human general transcription factor TFIIA: characterization of a cDNA encoding the small subunit and requirement for basal and activated transcription.

The human general transcription factor TFIIA is one of several factors involved in specific transcription by RNA polymerase II, possibly by regulating the activity of the TATA-binding subunit (TBP) of TFIID. TFIIA purified from HeLa extracts consists of 35-, 19-, and 12-kDa subunits. Here we describe the isolation of a cDNA clone (hTFIIA gamma) encoding the 12-kDa subunit. Using expression constructs derived from hTFIIA gamma and TFIIA alpha/beta (which encodes a 55-kDa precursor to the alpha and beta subunits of natural TFIIA), we have constructed a synthetic TFIIA with a polypeptide composition similar to that of natural TFIIA. The recombinant complex supports the formation of a DNA-TBP-TFIIA complex and mediates both basal and Gal4-VP16-activated transcription by RNA polymerase II in TFIIA-depleted nuclear extracts. In contrast, TFIIA has no effect on tRNA and 5S RNA transcription by RNA polymerase III in this system. We also present evidence that both the p55 and p12 recombinant subunits interact with TBP and that the basic region of TBP is critical for the TFIIA-dependent function of TBP in nuclear extracts.

Characterization of the highly conserved TFIIA small subunit from Drosophila melanogaster.

TFIIA is a general transcription factor that modulates class II transcription initiation in vitro by functionally and physically interacting with TFIID or the derived TATA-binding protein (TBP). TFIIA was previously purified from human, bovine, rat, and yeast sources and has recently been identified in association with TFIID in Drosophila. Here, we report the cloning of a cDNA encoding the 12.5-kDa subunit of TFIIA from Drosophila melanogaster (dTFIIA-S) and the identification of a partial dTFIIA-S gene in Drosophila virilis. The deduced amino acid sequence of dTFIIA-S indicates a high degree of homology to the small TFIIA subunit from yeast and to a partial TFIIA-S cDNA identified in rice. A hybrid TFIIA consisting of recombinant dTFIIA-S and the recombinant human TFIIA/alpha gene product mimics natural TFIIA activity in a TBP-dependent DNA binding assay. The promoter complex formed with this hybrid TFIIA depends upon the TATA element and can be efficiently incorporated into a higher order preinitiation complex upon addition of TFIIB. The presence of dTFIIA-S within the TBP-TFIIA-promoter complex was demonstrated using anti-dTFIIA-S antiserum. Finally, the ability of recombinant dTFIIA-S to reconstitute transcriptionally active TFIIA was demonstrated in a well defined human transcription system.

A single cDNA, hTFIIA/alpha, encodes both the p35 and p19 subunits of human TFIIA.

TFIIA is a transcription factor that, by interacting with the TATA-binding subunit (TBP) of TFIID, modulates transcription initiation by RNA polymerase II in vitro. By use of a mobility shift assay, TFIIA was purified from HeLa cells as a complex of 35-, 19-, and 12-kD subunits. Oligonucleotides were used to isolate a human cDNA clone, hTFIIA/alpha, which encodes a 55-kD protein with homology to the product of the yeast gene TOA1. The open reading frame of hTFIIA/alpha contains peptide sequences obtained from both the p35 and p19 subunits of natural human TFIIA, and thus encodes these two subunits. Consistent with this, antiserum raised against the 55-kD hTFIIA/alpha-encoded protein reacted with both the p35 and p19 subunits of natural TFIIA, and the recombinant protein could functionally replace those subunits in a mobility shift assay with renatured p12. An efficient affinity purification for natural human TFIIA was suggested by the sequence of the hTFIIA/alpha protein and demonstrated biochemically. Finally, transcription from the adenovirus major late promoter was greatly reduced in nuclear extracts depleted with anti-TFIIA/alpha serum and was restored to original levels by the readdition of purified human TFIIA.