Partial Proteolytic Mapping Research Articles

The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase.

Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI). The Q motif, consisting of nine amino acids (GFXXPXPIQ) with an invariant glutamine (Q) residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase.

Identification of the Nef-associated kinase as p21-activated kinase 2

The Nef protein of primate immunodeficiency viruses plays an important role in the pathogenesis of acquired immunodeficiency syndrome (AIDS) [1,2]. The interaction of Nef with the Nef-associated kinase (NAK) is one of the most conserved properties of different human and simian immunodeficiency virus (HIV and SIV) Nef alleles. The role of NAK association is currently not known but it has been implicated in enhanced viral infectivity in cell culture and in disease progression in SIV-infected macaques [3]. Previous studies have indicated that NAK shares many features with the p21-activated kinases (PAKs) [3], but the molecular identity of NAK has remained unknown. We have generated specific antisera against PAKs 1–3, and expressed these kinases individually as epitope-tagged proteins. By using these reagents in experiments involving partial proteolytic mapping, and exploiting the unique ability of PAK2 to serve as a caspase substrate, we have positively identified NAK as PAK2. Interestingly, although ectopic PAK2 overexpression efficiently replaced endogenous PAK2 from the complex with Nef, the total Nef-associated PAK2 activity was not increased, indicating the abundance of another cellular factor(s) as the limiting factor in Nef–PAK2 complex formation. Identification of NAK as PAK2 should now facilitate elucidation of its role as a mediator of the pathogenic effects of Nef.

Serine 257 phosphorylation regulates association of polyomavirus middle T antigen with 14-3-3 proteins.

Polyomavirus middle T antigen (MT) is phosphorylated on serine residues. Partial proteolytic mapping and Edman degradation identified serine 257 as a major site of phosphorylation. This was confirmed by site-directed mutagenesis. Isoelectric focusing of immunoprecipitated MT from transfected 293T cells showed that phosphorylation on wild-type MT occurred at near molar stoichiometry at S257. MT was previously shown to be associated with 14-3-3 proteins, which have been connected to cell cycle regulation and signaling. The association of 14-3-3 proteins with MT depended on the serine 257 phosphorylation site. This has been demonstrated by comparing wild-type and S257A mutant MTs expressed with transfected 293T cells or with Sf9 cells infected with recombinant baculoviruses. The 257 site is not critical for transformation of fibroblasts in vitro, since S257A and S257C mutant MTs retained the ability to form foci or colonies in agar. The tumor profile of a virus expressing S257C MT showed a striking deficiency in the induction of salivary gland tumors. The basis for this defect is uncertain. However, differences in activity for the wild type and mutant MT lacking the 14-3-3 binding site have been observed in transient reporter assays.

Peripheral blood mononuclear cells express antigens associated with multidrug resistance in a small cell lung cancer cell line : Krebes KA, Mirski SEL, Pross HF, Cole SPC. Cancer Research Laboratories, RM 331 Botterell Hall, Queen's University, Kingston, Ont K7L 3N6. Anticancer Res 1993;13:317–21

H69AR is a multidrug resistant small cell lung cancer (SCLC) cell line that does not overexpress P-glycoprotein, the plasma membrane drug efflux pump usually associated with this type of resistance. Monoclonal antibodies (MAbs) were previously raised against H69AR cells, and three of these MAbs, 2.54, 3.50, and 3.186, cross-reacted with peripheral blood mononuclear cells. T cells (CD3+), B cells (CD19+), NK cells (CD16+) and monocytes (CD14+) expressed each of the three antigens, but to differing degrees. Immunoprecipitation and partial proteolytic mapping experiments demonstrated that the antigens detected by MAbs 2.54 and 3.186 are identical in H69AR cells and PBMCs. SCLC cells are known to express many hematopoietic antigens; however, this is the first report of SCLC multidrug resistance-associated antigens being expressed on hematopoietic cells.

Protein kinase C modulates the catalytic activity of topoisomerase II by enhancing the rate of ATP hydrolysis: evidence for a common mechanism of regulation by phosphorylation

The catalytic activity of topoisomerase II is stimulated approximately 2-3-fold following phosphorylation by either casein kinase II or protein kinase C. A previous study [Corbett, A. H., DeVore, R. F., & Osheroff, N. (1992) J. Biol. Chem. 267, 20513-20518] demonstrated that casein kinase II regulates the activity of topoisomerase II by specifically enhancing the ability of the enzyme to hydrolyze its ATP cofactor. To determine whether other protein kinases use a similar mechanism to activate the enzyme, the effects of protein kinase C mediated phosphorylation on the individual steps of the topoisomerase II catalytic cycle were assessed. Modification stimulated rates of enzyme-mediated ATP hydrolysis approximately 2.7-fold, but had no effect on any reaction that preceded this step, including enzyme.DNA binding, pre- or poststrand passage DNA cleavage/religation, or the double-stranded DNA strand passage event. Furthermore, the activation of ATP hydrolysis was reversed following treatment of phosphorylated topoisomerase II with alkaline phosphatase. As determined by partial proteolytic mapping, the site(s) of protein kinase C modification was (were) localized to the 350 amino acid C-terminal regulatory domain of topoisomerase II within approximately 50 amino acids of the site(s) phosphorylated by casein kinase II. Finally, while protein kinase C and casein kinase II were able to modify the enzyme simultaneously, rates of ATP hydrolysis for doubly-modified topoisomerase II were comparable to those observed for the enzyme following phosphorylation by either individual kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

P105, the NF-kappa B p50 precursor protein, is one of the cellular proteins complexed with the v-Rel oncoprotein in transformed chicken spleen cells

Active NF-kappa B-like transcription complexes are multimers consisting of one or two members of a family of proteins related to the c-Rel proto-oncoprotein. We have isolated a chicken cDNA encoding p105, the precursor protein for the p50 subunit of NF-kappa B. Sequence analysis shows that chicken p105 is approximately 70% identical to the mouse and human p105 proteins, containing the Rel homology domain in its N-terminal 370 amino acids and several ankyrinlike repeats in the C-terminal portion of the protein. The Rel homology domain is particularly highly conserved between chicken and mammalian p50, and an in vitro-synthesized, truncated chicken p105 protein, containing sequences that correspond to the predicted p50 protein, bound to a consensus kappa B site in an electrophoretic mobility shift assay. In v-Rel-transformed chicken spleen cells, v-Rel is found in high-molecular-weight complexes which include cellular proteins of approximately 124 kDa (p124) and 115 kDa (p115). Here we report that in vitro-produced p105 comigrates with p124 from v-Rel-transformed spleen cells and that p105 and p124 appear to be identical by partial proteolytic mapping with V8 protease. Furthermore, both p105 and p50 can complex directly with v-Rel and chicken c-Rel in vitro. However, in vitro association with p105 by v-Rel does not necessarily correlate with transformation, since one nontransforming v-Rel mutant can associate with p105 in vitro.

The N-Myc oncoprotein is associated in vivo with the phosphoprotein Max(p20/22) in human neuroblastoma cells.

Proteins encoded by the proto-oncogenes c-myc, L-myc, and N-myc contain at their carboxy-terminus a tripartite segment comprising a basic DNA binding region (BR), a helix-loop-helix (HLH) and a leucine zipper motif (Zip), that are believed to be involved in DNA binding and protein-protein interaction. The N-Myc oncoprotein is overexpressed in certain human tumors that share neuroectodermal features due to amplification of the N-myc gene. Using a monoclonal antibody directed against an N-terminal epitope of the N-Myc protein in immunoprecipitations performed with extracts of neuroblastoma cells, two nuclear phosphoprotein, p20/22, forming a hetero-oligomeric complex with N-Myc are identified. Both proteins are phosphorylated by casein kinase II in vitro. By partial proteolytic maps we show that p20 and p22 are structurally related to each other and that p20 is identical with Max, a recently described in vitro binding partner of myc proteins. Time course experiments show the presence of the complex in cellular extracts immunoprecipitated within a 5 min interval after the preparation of the cell extract. While the expression of N-myc is restricted, expression of both Max(p20/22) and the murine homolog Myn(p20/22) was observed in cells of diverse human and murine embryonal lineages as detected by heterologous complex formation. By introduction of expression vectors containing the wild type N-myc gene or N-myc genes with in frame deletions or point mutations into recipient cells and subsequent immunoprecipitation of the resulting N-Myc proteins we show that the HLH-Zip region is essential to the formation of the N-Myc-p20/22 complex.

Post-translational modification of the tegument proteins (VP13 and VP14) of herpes simplex virus type 1 by glycosylation and phosphorylation.

VP13 and VP14, major tegument proteins of herpes simplex virus type 1 (HSV-1) and the products of the UL47 gene, have been shown by partial proteolytic mapping to have closely related protein sequences. These proteins are phosphorylated in virus-infected cells, but not in preparations of purified virus. They also contain O-linked oligosaccharide units which include beta-1,4-N-acetyl galactosamine residues, as demonstrated by the binding of Dolichos biflorus lectin. This modification was detected only in purified virus and appears to be restricted to VP13/14 and VP22, another HSV-1 tegument protein.

Isolation of the human cdk2 gene that encodes the cyclin A- and adenovirus E1A-associated p33 kinase.

Cyclins are regulatory subunits which associate with kinases to form complexes that control many of the important steps in cell-cycle progression. The best characterized of the cyclin-containing complexes is the association of cyclin B with the p34cdc2 kinase. The p34cdc2/cyclin B complex is required for the G2 to M transition (see refs 1-4 for review), but the physiological role of other cyclin complexes is unclear. Human cyclin A binds independently to two kinases, associating with either p34cdc2 or a related protein, p33 (refs 5-7). In adenovirus-transformed cells, the viral E1A oncoprotein seems to associate with p33/cyclin A but not with p34cdc2/cyclin A (B. Faha, M.M., L-H.T. and E.H., manuscript submitted). To isolate the gene for p33, we have cloned several novel human cdc2-related genes. The protein product of one of these genes, cdk2 (cyclin-dependent kinase 2), shares 65% sequence identity with p34cdc2 (ref. 8) and 89% identity with the Xenopus Eg-1 gene product. Immunochemical characterization and partial proteolytic mapping show that the cdk2 gene product is the cyclin A-associated p33. Immunoprecipitations of the p33cdk2 protein suggest that it can act as a protein kinase in vitro. As p33cdk2 is bound to cyclin A and is targeted by the viral E1A protein, we suggest that the p33cdk2/cyclin A complex has a unique role in cell-cycle regulation of vertebrate cells.

Features of the renal parathyroid hormone-parathyroid hormone-related protein receptor derived from structural studies of receptor fragments

Our earlier results indicated that the binding moiety of the renal PTH receptor is an 85 kD protein that is susceptible to proteolytic cleavage to a 70 kD form that supports high-affinity binding and Gs coupling, and to a 50-55 kD form that contains the ligand binding domain but does not couple to Gs. In the present study we used [125I]hPTHrP-(1-34)amide and a chemical cross-linking technique to discern the structural features of the intact 85 kD PTH/PTHrP receptor that are retained in the proteolyzed forms to "structurally map" the receptor. The results of lectin chromatography and endoglycosidase treatment show that the partially proteolyzed receptor forms retain the complex, N-linked glycans present on the intact receptor. This conclusion is further supported by the finding that wheat germ agglutinin was equally effective at competitively inhibiting specific [125I]hPTHrP-(1-34)A binding to the 70 kD form and the intact 85 kD receptor. Specific binding of [125I]hPTHrP-(1-34)A to the intact 85 kD receptor or to the 70 kD form was completely abolished by treatment with disulfide reducing agents, and both partially proteolyzed receptor forms (70- and 50 kD) were shown to retain the small (less than or equal to 14 kD) labeled fragment that is released from the intact receptor by disulfide reduction. Lectin chromatography and endoglycosidase treatment revealed that the less than or equal to 14 kD receptor component is not glycosylated. The less than or equal to 14 kD fragment does not contain a transmembrane spanning region, as its release from the membrane can be affected without detergent solubilization. Identical partial proteolytic maps of the receptor were obtained whether the receptor was covalently labeled with [125I]hPTHrP-(1-34)amide or [125I]bPTH-(1-34). These results suggest a model of the renal PTH/PTHrP receptor binding moiety as a single-chain protein in which the sites of glycosylation, ligand binding, and the functionally critical disulfide bonds are in extracellular domains near one end of the protein and the sites of proteolysis reside near the other end of the protein. These studies also provide further confirmation that PTH and PTHrP bind to a structurally indistinguishable renal receptor and validate the use of PTHrP as a ligand for studies designed to characterize and purify the PTH receptor.

NF-kappa B: a family of inducible and differentially expressed enhancer-binding proteins in human T cells.

A sensitive DNA-protein crosslinking approach has been used to characterize four inducible T-cell proteins (50 kDa, 55 kDa, 75 kDa, and 85 kDa) that specifically bind to kappa B enhancer elements. Partial proteolytic mapping revealed a distinct cleavage pattern for three of these proteins. These polypeptides are sequestered as inactive precursors in the cytosol of unstimulated T cells but can be converted into active forms in vivo by phorbol ester stimulation or in vitro by detergent treatment. The induction of these proteins by phorbol ester results in a strikingly biphasic pattern of nuclear expression with the 55-kDa and 75-kDa species appearing within minutes, whereas the 50-kDa and 85-kDa species appear only several hours after cellular stimulation. These data suggest that NF-kappa B-binding activity may not correspond to a single polypeptide but rather a family of at least four inducible and differentially regulated DNA-binding proteins that are expressed with distinct kinetics in human T lymphocytes.

Immunoglobulin Fc binding activity is associated with the mouse hepatitis virus E2 peplomer protein

Antigenic variation among murine coronaviruses is associated primarily with the surface peplomer protein E2 (180,000 Dal. E2 is responsible for attachment of the virus to the host cell, MHV-induced cell fusion, and eliciting neutralizing antibody. We report here the molecular mimicry between E2 and Fc γ receptor (FcγR). Molecular mimicry between E2 and FcyR may allow the escape of virus-infected cells from destruction by immunological mechanisms. Rabbit IgG, monoclonal rat IgG 1 and IgG 2b, monoclonal mouse IgG 2a and IgG 2b, and the rat anti-mouse FcγR monoclonal antibody 2.4132 immunoprecipitated from MHV-JHM-infected cells a polypeptide with a molecular mass identical to that immunoprecipitated by anti-E2 antibodies. F(ab′) 2 fragments of rabbit IgG did not immunoprecipitate any proteins from MHV-infected cells. All of these antibodies did not immunoprecipitate any proteins from uninfected cells. The anti-mouse FcyR monoclonal antibody 2.4132 immunoprecipitated from MHV-JHM-, MHV-3-, or MHV-A59-infected L-2 cells and 17CL-1 cells, or MHV-JHM-infected cultures of neonatal BALB/c brain cells, a protein with a molecular weight identical to that of MHV-JHM E2. The anti-FcyR monoclonal antibody did not immunoprecipitate any proteins from uninfected cells. Furthermore, the 2.4132 monoclonal antibody (mab), unrelated rat and mouse monoclonal antibodies, and a goat antiserum against E2, but not normal goat serum, immunoprecipitated a 75,000- to 77,000-Da molecule from uninfected WEHI-3 cells, a FcγR bearing cell line. Several lines of evidence demonstrated that the protein immunoprecipitated by the anti-FcγR mab from MHV-JHM-infected cells is the E2 glycoprotein: (1) Partial proteolytic maps obtained by Staphylococcus aureus V-8 protease treatment of the 180,000-Da proteins immunoprecipitated by the anti FcγR mab and the anti-E2 mab were identical. (2) Sequential immunoprecipitation experiments from MHV-JHM-infected cells revealed that the same polypeptide chain was recognized by the anti-E2 mab and by the anti-FcγR mab 2.41G2. (3) Actinomycin D did not influence the induction and expression of the 180,000-Da polypeptide chain that was immunoprecipitated by the anti-FcγR mab, demonstrating that this protein is of viral origin.

Regulation of casein kinase II activity by epidermal growth factor in human A-431 carcinoma cells

In order to characterize more fully the mechanism by which casein kinase II is regulated in mammalian cells, the effect of epidermal growth factor (EGF) on the activity of the kinase in human A-431 carcinoma cells was examined. Treatment of cells with EGF prior to lysis consistently resulted in a transient 4-fold increase in the activity of cytosolic casein kinase II. Activity rose sharply between 20 and 30 min, peaked at approximately 50 min, and returned to basal levels by approximately 120 min. Similar results were obtained using the casein kinase II specific peptide substrate, Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu, or DNA topoisomerase II (which is specifically modified by the kinase in vivo and serves as a high affinity substrate in vitro) as the phosphate acceptor in assays. Identification of casein kinase II as the stimulated activity was confirmed by partial proteolytic mapping and phosphoamino acid analysis of modified topoisomerase II, by inhibition at nanomolar levels of heparin or micromolar levels of nonradioactive GTP, and by the ability to employ radioactive GTP as a direct phosphate donor. The EGF stimulation of casein kinase II was dependent on the availability of intracellular (but not extracellular) calcium. In addition, hormonal action was modulated by calcium/phospholipid-dependent protein kinase (protein kinase C). Casein kinase II stimulation did not require an increase in the concentration of the kinase, protein synthesis, the continual presence of a small effector molecule, or a direct interaction with the EGF receptor/tyrosine kinase. In contrast, hormonal activation of the kinase was dependent on the phosphorylation of casein kinase II or a terminal stimulatory factor.

A Novel cDNA Extension Procedure: Isolation of Chicken Fatty Acid Synthase cDNA Clones

Abstract We have developed a simple and versatile cDNA extension method using lambda-exonuclease-generated single-stranded DNA as a primer. This plasmid-based cDNA extension method can be used to synthesize unidirectional extensions of the existing cDNA clones or subcloned fragments of the untranslated and exon regions of genomic DNA clones. The method is simple to use and involves no addition of linkers or tailing. We have successfully used this method to isolate 4.6 kilobase pairs of chicken fatty acid synthase cDNA clones, starting from the fragment of a genomic clone coding for the untranslated region of the fatty acid synthase mRNA. About 2.8 kilobase pairs of the cDNA coding for the chicken fatty acid synthase has been sequenced. The sequence has an open reading frame coding for 945 amino acids of the fatty acid synthase. In the sequence, we have identified the enoyl reductase, NADPH binding region, a putative beta-ketoacyl reductase region, and the entire sequences of acyl carrier protein and the thioesterase domains. The arrangement of these partial activities in this sequence confirms the arrangement of these activities as determined through partial proteolytic mapping studies. The amino acid sequence of chicken fatty acid synthase deduced from cDNA sequences shows a high degree of homology with the rat fatty acid synthase sequence, suggesting that these multifunctional proteins are conserved evolutionarily.

C-erbA encodes multiple proteins in chicken erythroid cells.

To identify and characterize the proteins encoded by the erbA proto-oncogene, we expressed the C-terminal region of v-erbA in a bacterial trpE expression vector system and used the fusion protein to prepare antiserum. The anti-trp-erbA serum recognized the P75gag-erbA protein encoded by avian erythroblastosis virus and specifically precipitated six highly related proteins ranging in size from 27 to 46 kilodaltons from chicken embryonic erythroid cells. In vitro translation of a chicken erbA cDNA produced essentially the same pattern of proteins. Partial proteolytic maps and antigenicity and kinetic analyses of the in vivo and in vitro proteins indicated that they are related and that the multiple bands are likely to arise from internal initiations within c-erbA to generate a nested set of proteins. All of the c-erbA proteins are predominantly associated with chicken erythroblast nuclei. However, Nonidet P-40 treatment resulted in extraction of the three smaller proteins, whereas the larger proteins were retained. During differentiation of erythroid cells in chicken embryos, we found maximal levels of c-erbA protein synthesis at days 7 to 8 of embryogenesis. By contrast, c-erbA mRNA levels remained essentially constant from days 5 to 12. Together, our results indicate that posttranscriptional or translational mechanisms are involved in regulation of c-erbA expression and in the complexity of its protein products.

C-erbA encodes multiple proteins in chicken erythroid cells.

To identify and characterize the proteins encoded by the erbA proto-oncogene, we expressed the C-terminal region of v-erbA in a bacterial trpE expression vector system and used the fusion protein to prepare antiserum. The anti-trp-erbA serum recognized the P75gag-erbA protein encoded by avian erythroblastosis virus and specifically precipitated six highly related proteins ranging in size from 27 to 46 kilodaltons from chicken embryonic erythroid cells. In vitro translation of a chicken erbA cDNA produced essentially the same pattern of proteins. Partial proteolytic maps and antigenicity and kinetic analyses of the in vivo and in vitro proteins indicated that they are related and that the multiple bands are likely to arise from internal initiations within c-erbA to generate a nested set of proteins. All of the c-erbA proteins are predominantly associated with chicken erythroblast nuclei. However, Nonidet P-40 treatment resulted in extraction of the three smaller proteins, whereas the larger proteins were retained. During differentiation of erythroid cells in chicken embryos, we found maximal levels of c-erbA protein synthesis at days 7 to 8 of embryogenesis. By contrast, c-erbA mRNA levels remained essentially constant from days 5 to 12. Together, our results indicate that posttranscriptional or translational mechanisms are involved in regulation of c-erbA expression and in the complexity of its protein products.

Phosphorylation of DNA topoisomerase II in vivo and in total homogenates of Drosophila Kc cells. The role of casein kinase II.

The phosphorylation of DNA topoisomerase II in Drosophila Kc tissue culture cells was characterized by in vivo labeling studies and in vitro studies that examined the modification of exogenous enzyme in total homogenates of these embryonic cells. Several lines of evidence identified casein kinase II as the kinase primarily responsible for phosphorylating DNA topoisomerase II. First, the only amino acyl residue modified in the enzyme was serine. Second, partial proteolytic maps of topoisomerase II which had been labeled with [32P]phosphate by Drosophila cells in vivo, by cell homogenates in vitro, or by purified casein kinase II were indistinguishable from one another. Third, phosphorylation in cell homogenates was inhibited by micrograms/ml concentrations of heparin, micromolar concentrations of nonradioactive GTP, or anti-Drosophila casein kinase II antiserum. Fourth, cell homogenates were able to employ [gamma-32P]GTP as a phosphate donor nearly as well as [gamma-32P]ATP. Although topoisomerase II was phosphorylated in homogenates under conditions that specifically stimulate protein kinase C, calcium/calmodulin-dependent protein kinase, or cAMP-dependent protein kinase, modification was always sensitive to anti-casein kinase II antiserum or heparin. Thus, under a variety of conditions, topoisomerase II appears to be phosphorylated primarily by casein kinase II in the Drosophila embryonic Kc cell system.

A Comparison between the Capsid Proteins and the Products of in vitro Translation of Three Strains of Potato Virus X

SUMMARY The capsid proteins of three potato virus X isolates that belong to different strain groups on the basis of their reactions with host resistance genes have been compared by means of SDS-PAGE and partial proteolysis mapping. Considerable differences were observed in both the migration rates in SDS-PAGE and the peptide patterns generated after partial digestion with V8 protease of the capsid proteins of the isolates. The high molecular weight proteins synthesized by in vitro translation of genomic RNAs extracted from the three isolates were electrophoretically indistinguishable and antigenically unrelated to capsid proteins.

Abundant expression of polyomavirus middle T antigen and dihydrofolate reductase in an adenovirus recombinant.

A modular gene with a cDNA encoding the polyomavirus middle T antigen positioned behind the adenovirus type 2 major late promoter and tripartite leader was substituted for the E1a region in an adenovirus vector. Permissive human cells infected with this recombinant produce middle T protein at levels as high as those of the most abundant late adenoviral proteins, e.g., hexon or fiber. This level represents at least a 40-fold increase over that observed in a polyomavirus lytic infection of murine cells. Partial proteolytic mapping showed that this protein has the same primary structure as middle T protein produced in polyomavirus-infected murine cells. The adenovirus recombinant-generated middle T protein exhibited in vitro kinase activity, although at an approximately 10-fold-lower specific activity than that of middle T protein from polyomavirus-infected murine cells. Comparison of the expression levels of this middle T antigen-containing adenovirus vector with a similar construction encoding dihydrofolate reductase suggested that the translation efficiency of the inserted gene was dependent upon the proximity of its initiation codon to the tripartite leader. We tested this possibility by comparing three dihydrofolate reductase recombinants among which the spacing between the initiation codon and tripartite leader varied from 188 to 36 nucleotides. The efficiency of expression of dihydrofolate reductase protein dramatically increased as this spacing was reduced.

Tightly bound nonprotamine proteins from ram sperm nuclei studied by one- and two-dimensional peptide mapping

The tightly bound proteins of ram sperm nuclei (TBSP) have been recovered as a fraction co-sedimenting with DNA after high salt-urea deprotamination of the nuclei. TBSP were studied by two-dimensional (2D)-tryptic peptide mapping and by one-dimensional (1D)-partial proteolysis mapping. The 2D maps revealed a strong homology among the proteins, irrespective of substantial differences in their molecular masses. This homology was supported also by the 1D-mapping data. The 2D-tryptic maps of TBSP were compared to those of lamb liver lamins but no apparent similarity was detected. TBSP were found to react positively to a test for the presence of carbohydrate residues, suggesting that these proteins are glycoproteins as established earlier for the lamins. The 2D maps of several proteins of seminal plasma origin, used as a control, displayed completely different peptide profiles.