Identification of polyol-responsive monoclonal antibodies for use in immunoaffinity chromatography.

One of the limitations of immunoaffinity chromatography as been that high-affinity antigen-antibody complexes are difficult to dissociate, often leading to inactivation of the protein product during elution from the immobilized antibody. As described in this unit, some antigen-antibody complexes can be dissociated in the presence of a combination of a low-molecular-weight polyhydroxylated compound (polyol) and a nonchaotropic salt. These conditions seem to be generally nondenaturing and, in some cases, even protein-stabilizing. This type of antibody is designated "polyol-responsive." These antibodies can be easily identified and isolated as monoclonal antibodies (MAbs) from a typical fusion, using standard hybridoma procedures. They have proven to be very valuable reagents for the immunoaffinity purification of active, labile, multi-subunit protein complexes.

CDK9 autophosphorylation regulates high-affinity binding of the human immunodeficiency virus type 1 tat-P-TEFb complex to TAR RNA.

Human immunodeficiency virus type 1 (HIV-1) Tat interacts with cyclin T1 (CycT1), a regulatory partner of CDK9 in the positive transcription elongation factor (P-TEFb) complex, and binds cooperatively with CycT1 to TAR RNA to recruit P-TEFb and promote transcription elongation. We show here that Tat also stimulates phosphorylation of affinity-purified core RNA polymerase II and glutathione S-transferase-C-terminal-domain substrates by CycT1-CDK9, but not CycH-CDK7, in vitro. Interestingly, incubation of recombinant Tat-P-TEFb complexes with ATP enhanced binding to TAR RNA dramatically, and the C-terminal half of CycT1 masked binding of Tat to TAR RNA in the absence of ATP. ATP incubation lead to autophosphorylation of CDK9 at multiple C-terminal Ser and Thr residues, and full-length CycT1 (amino acids 728) [CycT1(1-728)], but not truncated CycT1(1-303), was also phosphorylated by CDK9. P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of ATP, as did a C-terminal truncated CDK9 mutant that was catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins revealed that the 101-amino-acid SF2 HIV-1 Tat was unable to bind TAR with CycT1(1-303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restored binding of CycT1(1-303)-D167N-Tat, and rendered D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat-P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.

Architecture of RNA polymerase II and implications for the transcription mechanism.

A backbone model of a 10-subunit yeast RNA polymerase II has been derived from x-ray diffraction data extending to 3 angstroms resolution. All 10 subunits exhibit a high degree of identity with the corresponding human proteins, and 9 of the 10 subunits are conserved among the three eukaryotic RNA polymerases I, II, and III. Notable features of the model include a pair of jaws, formed by subunits Rpb1, Rpb5, and Rpb9, that appear to grip DNA downstream of the active center. A clamp on the DNA nearer the active center, formed by Rpb1, Rpb2, and Rpb6, may be locked in the closed position by RNA, accounting for the great stability of transcribing complexes. A pore in the protein complex beneath the active center may allow entry of substrates for polymerization and exit of the transcript during proofreading and passage through pause sites in the DNA.

Immunoaffinity purification of the RAP30 subunit of human transcription factor IIF.

RAP30, an RNA polymerase-associated protein (RAP) of approximately 30 kDa, is a component of the eukaryotic general transcription factor IIF (TFIIF). We have isolated a monoclonal antibody (MAb) that can be used to purify RAP30 under nondenaturing conditions. This MAb (designated 1RAP1) is a unique type of MAb that we have designated "polyol-responsive MAb." Polyol-responsive MAbs are high-affinity antibodies that release antigen in a buffer containing a low-molecular-weight polyhydroxylated compound (polyol) and a nonchaotropic salt. RAP30, contained on pET11d, was expressed in Escherichia coli by culturing and inducing protein expression at 26 degrees C. Under these conditions, approximately 50% of the RAP30 remains soluble. Inclusion bodies were removed from the cell lysate by centrifugation, the supernatant was treated with polyethyleneimine at 0.5 M NaCl to remove nucleic acids, and the soluble protein was applied directly to MAb-conjugated Sepharose. After extensive washing, RAP30 was eluted with buffer containing 0. 75 M ammonium sulfate and 40% propylene glycol. RAP30 produced by this procedure stimulates transcription from a minimal promoter. This is a rapid method for purifying unmodified RAP30 without renaturing the protein from inclusion bodies.

Yeast RNA polymerase II at 5 A resolution.

Appropriate treatment of X-ray diffraction from an unoriented 18-heavy atom cluster derivative of a yeast RNA polymerase II crystal gave significant phase information to 5 A resolution. The validity of the phases was shown by close similarity of a 6 A electron density map to a 16 A molecular envelope of the polymerase from electron crystallography. Comparison of the 6 A X-ray map with results of electron crystallography of a paused transcription elongation complex suggests functional roles for two mobile protein domains: the tip of a flexible arm forms a downstream DNA clamp; and a hinged domain may serve as an RNA clamp, enclosing the transcript from about 8-18 residues upstream of the 3'-end in a tunnel.

Modified adductor muscle transfer in cerebral palsy.

Eighty-five patients with cerebral palsy had modified adductor muscle transfers. A study of associated patient characteristics suggests that comparing adductor transfer with adductor release using postoperative radiographs, need for subsequent surgery, or postoperative motor skills is flawed by multiple variables. Adductor release and adductor transfer are best compared by measuring the abduction obtained at surgery and maintained over time. A follow-up of 141 modified adductor transfers with no prior or concomitant hip surgery demonstrated an averaged initial improvement in abduction of 43 degrees and maintenance of abduction with a low incidence of recurrence.

Identification of the epitope for a highly cross-reactive monoclonal antibody on the major sigma factor of bacterial RNA polymerase.

A highly cross-reactive monoclonal antibody (MAb), 2G10, was found to react in a conserved region of Escherichia coli RNA polymerase sigma70. The epitope was localized to amino acids 470 to 486, which included part of conserved region 3.1. The epitope for MAb 3D3, a MAb which maps close to the 2G10 epitope, was also determined.

The ABL-MYC retrovirus generates antigen-specific plasmacytomas by in vitro infection of activated B lymphocytes from spleen and other murine lymphoid organs.

ABL-MYC is a recombinant retrovirus that constitutively expresses the v-abl and c-myc oncogenes. When used to infect immunized mice this virus rapidly and efficiently induces plasmacytomas of which an unusually high percentage secrete antigen (Ag)-specific monoclonal antibodies. These findings suggested that ABL-MYC targets Ag-stimulated B cells for transformation and that infection of lymphoid cells in vitro might be a useful, alternative method for generating monoclonal, Ag-specific plasmacytomas (ASPCTs). Therefore, we used helper virus-free ABL-MYC to infect suspensions of cells from spleens and other lymphoid organs from mice that had been immunized with a variety of Ags and transplanted them into naive mice. The results show that ABL-MYC preferentially transforms splenocytes that are Ag-reactive. They also demonstrate that ASPCTs can be produced by in vitro infection of cell suspensions from the spleen, lymph nodes and Peyer's patches of mice that had been immunized intraperitoneally with sheep red blood cells, Escherichia coli core RNA polymerase or Epstein-Barr virus gp340 protein or immunized orally with live Giardia lamblia parasites. The ASPCTs usually consisted of one to three colnes, secreted antibodies that were quantitatively and qualitatively similar to those obtained from hybridomas, and could continue to secrete Ag-reactive antibody over eight transplant generations.

A novel collection of accessory factors associated with yeast RNA polymerase II.

A relatively simple subset of general transcription factors is sufficient for transcript initiation by RNA polymerase II. However, a recently identified "holoenzyme" contains additional accessory proteins required for mediating signals from some activators (Y-J. Kim et al., 1994, Cell 77, 599-608; A. Koleske and R. Young, 1994, Nature 368, 466-469). By immobilizing RNA polymerase II and associated proteins (RAPs) from a transcriptionally active yeast extract, we have identified a novel collection of proteins distinct from those found in the holoenzyme. The eluted RAP fraction did not contain the holoenzyme components Srb2,4,5 + 6p, Gal11p, or Sug1p, but did include the known transcription factors TFIIB and TFIIS and the three subunits of yeast TFIIF (Ssu71p/Tfg1p, Tfg2p, and Anc1p/Tfg3p). Also isolated as RAPs are two proteins (Cdc73p and Paf1p) with interesting connections to gene expression. Mutations in CDC73 and PAF1 affect cell growth and the abundance of transcripts from a subset of yeast genes (X. Shi et al., Mol. Cell. Biol., 1996 16, 669-676). The RAP fraction may therefore define one or more functional forms of RNA polymerase II distinct from the activator-mediating holoenzyme.

Testosterone effects on vasotocinergic innervation of sexually dimorphic medial preoptic nucleus and lateral septum during aging in male quail.

Vasotocin fibers are known to innervate regions important in the regulation of sexual behavior and neuroendocrine systems in quail. In this experiment, vasotocinergic innervation of the lateral septum and of the sexually dimorphic medial preoptic nucleus was studied during reproductive aging relative to sexual behavior or following testosterone (T). There were 4 groups of male Japanese quail (Coturnix japonica) studied: adult reproductive (6 month, n = 4), photoregressed adult (n = 5), old senescent (36 month, n = 4), and old testosterone-treated (n = 5). Immunocytochemistry for vasotocin (VT) was performed on serial sections and quantification of the density of VT-positive fibers was performed by image analysis. Results showed a highly significant decrease in VT-immunocytochemical staining in photoregressed and in old senescent males; whereas T-treatment in old males was associated with recovery of VT-immunocyto-chemical staining, comparable to the adult reproductive male. Previous experiments have shown that T treatment restimulates sexual behavior in senescent males similar to the recovery of sexual behavior in T-treated castrates. These results indicate that the VT system may be associated with the behavioral recovery observed in senescent T-treated males.

Accessibility of epitopes on human transcription factor IIB in the native protein and in a complex with DNA.

Transcription factor IIB (TFIIB) plays a central role in the assembly of the RNA polymerase II initiation complex. Monoclonal antibodies (mAbs) that react with human TFIIB were prepared and used as probes to identify portions of TFIIB that are accessible when the factor is in solution and when it is contained in a complex with DNA. Seven mAbs were examined and were mapped to three regions of the TFIIB molecule. Only the mAbs that mapped to residues 52-105 inhibited transcription, immunoprecipitated recombinant TFIIB and TFIIB from HeLa cell nuclear extract (NE), and supershifted a complex containing TFIIB, the TATA-binding protein, and DNA. The mAbs that mapped to residues 1-51 and the mAb that mapped to residues 106-316 did not show activity in the functional assays, with the exception of the far N-terminal mAbs (residues 1-51), which immunoprecipitated recombinant TFIIB, but not TFIIB from HeLa cell NE. These data indicate that the region containing residues 52-105 is exposed in solution and when TFIIB is part of the preinitiation complex and that some far N-terminal epitopes are accessible on the purified protein, but become blocked when TFIIB is in HeLa cell NE or in the preinitiation complex.

Purification of recombinant human transcription factor IIB by immunoaffinity chromatography.

The human RNA polymerase II transcription factor IIB (TFIIB) was purified from a bacterial expression system by immunoaffinity chromatography. Mouse monoclonal antibodies (MAbs) were prepared that react with TFIIB. A modified enzyme-linked immunosorbent assay was used to screen for MAbs that release the antigen in the presence of a low molecular weight polyhydroxylated compound and a nonchaotropic salt (polyol-responsive MAbs). One polyol-responsive MAb (designated IIB8) was purified by chromatography on protein A and conjugated to cyanogen bromide-activated Sepharose 4B. Escherichia coli strain BL21 (DE3) containing the pLysS plasmid was transformed with the human TFIIB gene contained in the pET11a vector (phIIB). After induction with IPTG, the cells were harvested and lysed. The lysate was treated with 0.5% polyethyleneimine and centrifuged. The supernatant fluid was applied to the IIB8-Sepharose. After extensive washing, the TFIIB was eluted with Tris-EDTA buffer containing 0.75 M ammonium sulfate and 40% propylene glycol. The purified TFIIB was active when added back to TFIIB-depleted HeLa nuclear extract and when used in the IgH minimal promoter system. This method will be useful for the rapid purification of TFIIB mutants and for the purification of large amounts of highly purified TFIIB for biochemical studies. In addition, this procedure establishes the general applicability of the use of polyol-responsive MAbs for the rapid, gentle purification of labile proteins.

In vitro functional characterization of overproduced Escherichia coli katF/rpoS gene product.

The katF/rpoS gene product (sigma s), a central regulator of stationary-phase gene expression in Escherichia coli, has been purified from an overproducing strain. sigma s was used as an immunogen for the production of monoclonal antibodies. Previous sequence analysis of sigma s strongly indicated homology to the sigma factor family. We show biochemically in this paper that sigma s is a sigma factor. This protein can bind to core RNA polymerase (E), and this binding can be competed effectively by the major E. coli transcription initiation factor, sigma 70. Immunopurified sigma s holoenzyme (E sigma s) transcribes the promoters of the bolAp1 gene and the xthA gene. Interestingly, both promoters can also be transcribed by sigma 70 holoenzyme (E sigma 70).

Isolation and characterization of a polyol-responsive monoclonal antibody useful for gentle purification of Escherichia coli RNA polymerase.

A modified enzyme-linked immunosorbent assay (ELISA) was used to screen monoclonal antibodies (MAbs) that react with Escherichia coli RNA polymerase for the ability to release the RNA polymerase in the presence of a low molecular weight polyhydroxylated compound (polyol) and a non-chaotropic salt. This assay, termed the ELISA-elution assay, identified 19 presumptive "polyol-responsive" MAbs out of a total of 218 antigen-specific MAbs screened. One of these MAbs, designated NT73, was examined in detail for the ability to release the antigen in response to various combinations of polyol and salt. Using NT73 conjugated to Sepharose, highly active RNA polymerase could be prepared rapidly by a single immunoaffinity chromatography step, replacing two lengthy chromatographic steps in our conventional purification procedure. Because NT73 reacts with the beta' subunit of RNA polymerase, a mixture of the core polymerase and holoenzyme was recovered from the immunoaffinity column. The holoenzyme (E sigma 70) could be separated from the core polymerase by subsequent chromatography on a Mono Q column. This demonstrates that polyol-responsive MAbs can be easily identified and characterized by the ELISA-elution assay. The use of polyol-responsive MAbs provides a means of adapting immunoaffinity chromatography to the purification of labile proteins.

The HIP1 initiator element plays a role in determining the in vitro requirement of the dihydrofolate reductase gene promoter for the C-terminal domain of RNA polymerase II.

We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.

Purification and characterization of proximal sequence element-binding protein 1, a transcription activating protein related to Ku and TREF that binds the proximal sequence element of the human U1 promoter.

The promoter structure of the known small nuclear RNA (snRNA) genes contains two major effectors of transcriptional activity, a proximal sequence element (PSE) and a distal sequence element (DSE). In previous work, methidiumpropyl-EDTA-Fe(II) footprinting was used to demonstrate the existence in human placental extracts of a protein producing footprints within the PSE and the DSE of the human U1 snRNA gene. This protein (PSE1) has now been purified to homogeneity from both human placental extract and K562 cell nuclear extract. PSE1 consists of two subunits, an alpha subunit with an apparent molecular mass of 83 kDa, and a beta subunit with an apparent molecular mass of 73 kDa in K562 nuclear extracts and 63 kDa in placental extracts. Footprinting and UV cross-linking assays indicate that purified PSE1 binds to the PSE and DSE of the U1 gene. Monoclonal antibodies were prepared which specifically recognize the individual subunits of PSE1. PSE1 is immunologically similar to and shares amino acid sequence with a protein (TREF) which binds the human transferrin receptor (HTFR) promoter. An in vitro transcription system was established for a template consisting of a minimal HTFR promoter placed upstream of the human U1 snRNA-coding region and shown by immunodepletion/addback experiments to specifically require PSE1. Transcription from the adenovirus 2 major late promoter was unaffected in these experiments. This result supports a functional role of PSE1 as a transcriptional activating protein, but its role in transcription of snRNA genes remains to be established. PSE1 also has an immunological relationship to and shares amino acid sequence with the p70 and p86 subunits of the human Ku autoantigen. Ku, PSE1, and TREF may thus be identical proteins or members of a family of heterodimeric proteins consisting of related subunits. Our results support earlier proposals that Ku may be a transcriptional activator.

Chlamydia trachomatis RNA polymerase major sigma subunit. Sequence and structural comparison of conserved and unique regions with Escherichia coli sigma 70 and Bacillus subtilis sigma 43.

We identified and sequenced the gene for the Chlamydia trachomatis RNA polymerase major sigma subunit. The gene encodes a 66,141-dalton protein (sigma 66), intermediate in size between the major sigma subunits of Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43). The C. trachomatis sigma 66 subunit had extensive amino acid homology with the sigma 70 and sigma 43. The sigma subunit regions purportedly involved in core enzyme binding and DNA promoter recognition were also highly conserved, despite the lack of a DNA promoter consensus sequence between E. coli and C. trachomatis promoters and the inability of E. coli holoenzyme to specifically transcribe chlamydial genes. Compared with E. coli sigma 70, there were some major differences in the chlamydial sigma 66 sequence, including a gap of 63 amino acids and an additional 16 amino acids at the carboxyl terminus, which may play some role in modifying the sigma-DNA interaction, such that a promoter sequence unique to C. trachomatis is recognized. Monoclonal antibodies specific for E. coli sigma 70 were used to probe for homologous structures between sigma 70 and sigma 66; only one of seven antibodies bound specifically to sigma 66, suggesting minimal conservation of antigenic sites. The chlamydial sigma 66 was present in elementary bodies and was expressed throughout the developmental cycle, which implied that this gene encodes the major vegetative sigma subunit. Because the ability to study the genetics of C. trachomatis is currently limited, this work provides a tool for more detailed study of chlamydial promoter structure and of coordinate gene expression during the developmental cycle.

Purification of eukaryotic RNA polymerase II by immunoaffinity chromatography. Elution of active enzyme with protein stabilizing agents from a polyol-responsive monoclonal antibody.

Active eukaryotic RNA polymerase II (RNAP II) was purified by immunoaffinity chromatography, using a monoclonal antibody (mAb) that reacts with the highly conserved heptapeptide repeat of the largest subunit. This mAb (designated SWG16) was conjugated to CNBr-activated Sepharose and used to purify RNAP II from wheat germ and calf thymus. The subunit composition of the immunoaffinity-purified enzyme was essentially the same as RNAP II purified by conventional chromatography except that it contained only the form with the unproteolyzed largest subunit. Active enzyme could be eluted from the SWG16-Sepharose, at pH 7.9, with combinations of low molecular weight polyols and nonchaotropic salts. The superior eluting procedure used combinations of ethylene glycol (30-40%) and ammonium sulfate (0.5-0.75 M). Active enzyme also could be eluted with a synthetic peptide containing four repeats of the heptapeptide; however, the peptide was not as effective as the polyol and salt combinations for eluting the enzyme. This mAb should be useful for purifying RNAP II from many eukaryotic species. Because the elution of enzyme from the immunoadsorbent seems to be dependent upon the presence of a polyol, this antibody is referred to as a "polyol-responsive mAb." A procedure that helps to identify a polyol-responsive mAb and to optimize the eluting conditions is described. Polyol-responsive mAbs might have broad applicability to the purification of many labile enzymes by immunoaffinity chromatography.

Purification and lipid-layer crystallization of yeast RNA polymerase II.

Yeast RNA polymerase II was purified to homogeneity by a rapid procedure involving immunoaffinity chromatography. The purified enzyme contained 10 subunits, as reported for conventional preparations, but with no detectable proteolysis of the largest subunit. In assays of initiation of transcription at the yeast CYC1 promoter, the enzyme complemented the deficiency of an extract from a strain that produces a temperature-sensitive polymerase II. Mammalian RNA polymerase II was inactive in this initiation assay. The purified yeast enzyme formed two-dimensional crystals on positively charged lipid layers, as previously found for Escherichia coli RNA polymerase holoenzyme. Image analysis of electron micrographs of crystals in negative stain, which diffracted to about 30-A resolution, showed protein densities of dimensions consistent with those of single polymerase molecules.