Characterization of peripheral blood T-lymphocytes transduced with HTLV-I Tax mutants with different trans-activating phenotypes.

Tax1, a transcriptional trans-activator of the Human T-cell leukemia virus type I (HTLV-I), induces the expression of many cellular genes through interaction with at least three distinct cellular transcription factors; CREB/ATF, NF-kappaB, and SRF. This Tax1-induced activation of cellular genes is considered to be a critical event in T-cell transformation by HTLV-I. To elucidate the role of each Tax1-inducible transcriptional pathway in T-cell transformation, we introduced Tax1 mutants with different trans-activating phenotypes into peripheral blood lymphocytes (PBL) by retroviral vectors. Analysis of these PBLs revealed that activation of the NF-kappaB pathway is sufficient to promote the growth response to IL-2. However, for the clonal expansion of CD4+ T-cells, which is a characteristic result of HTLV-I infection, activation of the CREB/ATF and SRF pathways is also required.

Src-homology domain 2 is responsible for transcriptional suppression induced by expression of Lck.

Overexpression of Lck was shown, by our previous study, to suppress gene transcription from various viral and cellular promoters. The suppression of transcription from human T-cell leukemia virus promoter by Lck was independent of the presence of the enhancer core sequences within the long terminal repeat. The suppression of transcription was observed with Lck mutants that had either diminished or enhanced tyrosine-kinase activity. A mutant lacking the myristylation site also suppressed transcription. From the analysis with various deletion mutants of Lck, it was suggested that Src-homology domain 2 (SH2) is both necessary and sufficient for the suppression of transcription. A similar effect was also observed with the SH2 domain of the v-src gene. Thus, overexpression of Lck could suppress gene expression through a unique function of the SH2 domain.

Murine retroviral vectors expressing the tax1 gene of human T-cell leukemia virus type 1.

Murine retroviral vectors which express the tax1 gene of human T-cell leukemia virus type 1 (HTLV-1) have been constructed. A significant increase in virus titer was achieved by inserting a portion of the gag region of Moloney murine leukemia virus. Using these vectors, tax1 was stably introduced into primary human T-cells derived from peripheral blood lymphocytes. Expression of the functional tax1 in infected cells was confirmed by trans-activation of HTLV-1 long terminal repeat-directed transcription. These vectors provide a useful means of investigating the function of tax1 in natural target cells for HTLV-1.

Cloning of a cDNA encoding a DNA-binding protein TAXREB302 that is specific for the tax-responsive enhancer of HTLV-I.

The transcriptional activator, Tax, of human T-cell leukemia virus (HTLV-I) has been considered to interact with cellular proteins to act on target enhancer motifs. Using oligodeoxyribonucleotides containing the tax-responsive element (TAXRE) of the HTLV-I enhancer, we have cloned multiple cDNAs coding for TAXRE-binding proteins (TAXREB), and determined the cDNA and the deduced 200-amino-acid sequences for TAXREB302. The recombinant protein binds to the enhancer DNA by specific interaction to the CRE-like sequence. A single 1.8-kb species of mRNA was detected in cultured cells, as well as in normal human tissues, especially brain and skeletal muscle. The 22-kDa native protein was detected in the cultured-cell lysate by immunoblotting analysis. TAXREB302 does not have structural features common to the CRE-binding protein or activating transcription factor (CREB/ATF) family, but has homology to chicken erythroid transcription factor (Eryf1 or GATA-1), suggesting a possible protein-protein interaction.

An antigenic structure of the trans-activator protein encoded by human T-cell leukemia virus type-I (HTLV-I), as defined by a panel of monoclonal antibodies.

In order to study an antigenic structure of the trans-activator protein encoded by human T-cell leukemia virus type-I (HTLV-I), tax1 antigen, we generated and characterized a panel of rat anti-tax1 monoclonal antibodies (MAbs) designated WATM-1, WATM-2, WATM-3, and WATM-4. These MAbs were derived from WKA rats immunized with HTLV-I-transformed (HTLV-I+) syngeneic T cells. Immunoblot assays showed that: (1) All the MAbs reacted with the tax1 antigen in HTLV-I+ cell lines and a recombinant tax1 antigen, PX141 (containing entire tax1 polypeptide); (2) WATM-3 and WATM-4, but not WATM-1 or WATM-2, reacted with a truncated tax1 antigen, XD59 (tax1 amino acids 180-338); (3) None of them reacted with another truncated tax1 antigen, XD128 (tax1 amino acids 1-47 and 286-353); and (4) each of the four MAbs had different reactivity with tax1-related antigens in the range 38-41 kDa expressed in simian cell lines infected with various HTLV-I-related simian retroviruses (STLV-I). None of the MAbs reacted with HTLV-II tax antigen. Human sera containing anti-tax1 antibodies interfered specifically with the antigen-specific binding of all the MAbs. These results suggest that the present rat MAbs are directed against various epitopes on the tax1 antigen. An antigenic structure of the tax1 antigen deduced from reactivity of a panel of anti-tax1 MAbs including the present rat MAbs is discussed.

Isolation of a cDNA clone encoding DNA-binding protein (TAXREB107) that binds specifically to domain C of the tax-responsive enhancer element in the long terminal repeat of human T-cell leukemia virus type I.

Five cDNA clones for TAXREB proteins that bind to the tax-responsive enhancer element of human T-cell leukemia virus type I (HTLV-I) were isolated from a Jurkat cell cDNA library. The beta-galactosidase fusion proteins of three of these clones specifically recognized the domain C within the enhancer. One of the three cDNAs, encoding TAXREB107, contained an open reading frame with 288 amino acid residues. RNA blot analysis showed that the level of mRNA for TAXREB107 increased transiently in Jurkat cells on treatment with TPA. Immunoblot analysis showed that polyclonal antibody against TAXREB107 specifically recognized a 34-kD protein in Jurkat cells. TAXREB107 may participate in tax-mediated trans-activation of transcription.

Expression of HTLV-I envelope protein fused to hydrophobic amino-terminal peptide of baculovirus polyhedrin in insect cells and its application for serological assays.

The envelope of human T-cell leukemia virus type I (HTLV-I) consists of two glycoproteins gp46 and p20E. Recombinant envelope proteins were produced by using an expression vector derived from insect baculovirus, Bombyx mori nuclear polyhedrosis virus. Polyhedrin fusion proteins C182, N147, and N287 contained whole region p20E, C-terminal half of gp46, and almost whole region gp46, respectively. N147 and N287 were suggested to be processed forms resulting from internal cleavage by cellular enzymes. In cultured cells and the insect larvae, C182 and N147 were produced abundantly enough to be purified to homogeneity; however, N287 was produced poorly and not purified. The purified proteins were recognized by HTLV-I-infected human sera and shown to be highly specific antigens for blood screening systems.

Evaluation of enzyme immunoassay using a recombinant envelope protein expressed in insect cells for serological confirmation of HTLV-I infection.

A recombinant human T-lymphotropic virus type I (HTLV-I) envelope protein expressed in insect cells using a recombinant baculovirus was employed as the antigen in an enzyme immunoassay (renvEIA). Peripheral blood samples were obtained from asymptomatic carriers or healthy individuals. Plasma was tested for HTLV-I antibody by renvEIA, particle agglutination, and Western immunoblot (WB), and lymphocyte DNA was tested for HTLV-I proviral DNA amplification by polymerase chain reaction (PCR). Of 61 people aged 9 months or older, 23 were positive (gag+, env+) and 19 others were in the "indeterminate" category (gag+, env-) when their WB results were interpreted according to the WHO-proposed criteria. Thirty-seven cases, including all of the WB+ cases and 14 of 19 WB indeterminate cases, were positive by renvEIA. In 34 of 37 renvEIA-positive cases, the presence of long terminal repeat (LTR) and tax/rex region of HTLV-I proviral DNA was detected by polymerase chain reaction (PCR) and following Southern blot hybridization. Thus, renvEIA would be a useful supplemental assay to confirm the presence of HTLV-I antibody in HTLV-I asymptomatic carriers.

The tomato mosaic tobamovirus movement protein interacts with a putative transcriptional coactivator KELP.

Viral movement through plasmodesmata in host plants likely depends on the interaction between virus-encoded movement protein (MP) and host proteins. In order to search for MP-interacting protein (MIP), we carried out far-western screening of a Brassica campestris cDNA library using a recombinant MP of tomato mosaic tobamovirus (ToMV) as a probe. One of the positive clones, designated MIP102, was found to be a putative orthologue for a transcriptional coactivator KELP of Arabidopsis thaliana. In vitro analysis with recombinant proteins revealed that ToMV MP could bind to KELP proteins that are derived from different plant species. At least 31 amino acids from the carboxyl-terminus of ToMV MP were dispensable for the interaction with KELP. Other MPs, derived from crucifer tobamovirus CTMV-W and cucumber mosaic cucumovirus, also exhibited comparable binding abilities. This suggests that these MPs could commonly interact with KELP, possibly to modulate the host gene expression.

Relationship between deficiency of phosphoglucose isomerase in Coprinus macrorhizus and fruiting body formation.

A mutant (pgi) of Coprinus macrorhizus deficient in phosphoglucose isomerase did not grow on fructose and grew poorly on glucose. The pgi mutation inhibited the formation of monokaryotic and dikaryotic fruiting bodies.

Sequence of the small subunit of yeast carbamyl phosphate synthetase and identification of its catalytic domain.

The yeast gene CPA1 coding for the small subunit of arginine-specific carbamyl phosphate synthetase has been cloned by complementation of a cpa 1 mutant with a plasmid library of total yeast chromosomal DNA. Two of the plasmids, pJL113/ST4 and pJL113/ST15, contain DNA inserts in opposite orientations with overlapping sequences of 2.6 kilobases. The nucleotide sequence of a 2.2-kilobase region of the DNA insert carrying the CPA1 gene has been determined. The CPA1 gene has been identified to be 1233 nucleotides long and to code for a polypeptide of 411 amino acids with a calculated molecular weight of 45,358. The amino acid sequence encoded in CPA1 is homologous to the recently determined sequence of the small subunit of Escherichia coli carbamyl phosphate synthetase (Piette, J., Nyunoya, H., Lusty, C.J., Cunin, R., Weyens, G., Crabeel, M., Charlier, D., Glandsdorff, N., and Pierard, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4134-4138) over the entire length of the polypeptide chain. Comparison of the amino acid sequences of the small subunits of yeast and E. coli carbamyl phosphate synthetases to the sequences of Component II of anthranilate and p-aminobenzoate synthases suggests that these amidotransferases are evolutionarily related. The most highly conserved region of the yeast and E. coli enzymes includes a cysteine residue previously found to be at the active site of Pseudomonas putida anthranilate synthase Component II (Kawamura, M., Keim, P.S., Goto, Y., Zalkin, H., and Heinrikson, R.L. (1978) J. Biol. Chem. 253, 4659-4668). Based on the observed homologies in the primary sequences of the other amidotransferases examined, we propose a 13-amino acid long sequence to be part of the catalytic domain of this class of enzymes.

The carB gene of Escherichia coli: a duplicated gene coding for the large subunit of carbamoyl-phosphate synthetase.

Previous genetic and biochemical studies indicate that the carB gene of Escherichia coli codes for the large subunit of carbamoyl-phosphate synthetase (EC 6.3.5.5). We have determined the nucleotide sequence of a 4-kilobase-pair cloned fragment of E. coli DNA with genetic determinants for carB. The DNA sequence is a 3,219-nucleotide-long reading frame. The polypeptide encoded by this reading frame has been verified to be the large subunit of carbamoyl-phosphate synthetase. The gene product is similar to the large subunit in its molecular weight, amino acid composition and amino-terminal residue, and carboxyl-terminal sequence. The amino acid sequence derived from the nucleotide sequence shows a highly significant homology between the amino- and carboxyl-terminal halves of the protein. We propose that the carB gene was formed by an internal duplication of a smaller ancestral gene.

The gene coding for carbamoyl-phosphate synthetase I was formed by fusion of an ancestral glutaminase gene and a synthetase gene.

A near full-length cDNA copy of rat carbamoyl-phosphate synthetase I (EC 6.3.4.16) mRNA has been cloned. The cDNA insert in the recombinant plasmid pHN234 is 5.3 kilobases long. Analysis of the sequence coding for carbamoyl-phosphate synthetase I indicates that the gene has arisen from a fusion of two ancestral genes: one homologous to Escherichia coli carA, coding for a glutaminase subunit, and the second homologous to the carB gene that codes for the synthetase subunit. A short amino acid sequence previously proposed to be part of the active site involved in glutamine amide nitrogen transfer in the E. coli and yeast carbamoyl-phosphate synthetases (EC 6.3.5.5) is also present in the rat enzyme. In the mammalian enzyme, however, the glutaminase domain lacks a cysteine residue previously shown to interact with glutamine. The cysteine is replaced by a serine residue. This substitution could, in part, account for the inability of mammalian carbamoyl-phosphate synthetase I to catalyze the hydrolysis of glutamine to glutamic acid and ammonia.

Catalytic domains of carbamyl phosphate synthetase. Glutamine-hydrolyzing site of Escherichia coli carbamyl phosphate synthetase.

We present evidence that cysteine 269 of the small subunit of Escherichia coli carbamyl phosphate synthetase is essential for the hydrolysis of glutamine. When cysteine 269 is replaced with glycine or with serine by site-directed mutagenesis of the carA gene, the resulting enzymes are unable to catalyze carbamyl phosphate synthesis with glutamine as nitrogen donor. Even though the glycine 269, and particularly the serine 269 enzyme bind significant amounts of glutamine, neither glycine 269 nor serine 269 can hydrolyze glutamine. The mutations at cysteine 269 do not affect carbamyl phosphate synthesis with NH3 as substrate. The NH3-dependent activity of the mutant enzymes was equal to that of wild-type. Measurements of Km indicate that the enzyme uses unionized NH3 rather than ammonium ion as substrate. The apparent Km for NH3 of the wild-type enzyme is calculated to be about 5 mM, independent of pH. The substitution of cysteine 269 with glycine or with serine results in a decrease of the apparent Km value for NH3 from 5 mM with the wild-type to 3.9 mM with the glycine, and 2.9 mM with the serine enzyme. Neither the glycine nor the serine mutation at position 269 affects the ability of the enzyme to catalyze ATP synthesis from ADP and carbamyl phosphate. Allosteric properties of the large subunit are also unaffected. However, substitution of cysteine 269 with glycine or with serine causes an 8- and 18-fold stimulation of HCO-3 -dependent ATPase activity, respectively. The increase in ATPase activity and the decrease in apparent Km for NH3 provide additional evidence for an interaction of the glutamine binding domain of the small subunit with one of the two known ATP sites of the large subunit.

The molecular characterization and in situ expression pattern of pea SCARECROW gene.

Certain mutants of shoot gravitropism were reported to be ascribed to the SCR and SHR loci in Arabidopsis thaliana. The SCR gene was known to regulate the development of endodermis cells that are responsible for sensing gravity in a shoot. With the aim of elucidating the molecular mechanism for gravitropic responses in pea seedlings, we have isolated a putative pea SCR ortholog from a shoot cDNA library. Analyses of the cDNA clones revealed the structure of a full-length ORF coding for 819 amino acid residues. A remarkable feature of pea SCR protein was the presence of asparagine stretches at the N-terminal transcriptional activation domain, which was distinct from the occurrence of glutamine or alanine stretches in the Arabidopsis or maize SCR. A Northern blot analysis revealed a single 3.2-kb pea SCR transcript in addition to a closely related 2.5-kb transcript. Our in situ hybridization data indicated that pea SCR mRNA accumulated in the shoot apical meristem, leaf primordia and a root single cell layer corresponding to the endodermis. The expression patterns were similar to those reported for A. thaliana and Zea mays, suggesting that SCR may be functionally conserved among plants and involved in the differentiation of the endodermis.

In vivo synthesis of carbamyl phosphate from NH3 by the large subunit of Escherichia coli carbamyl phosphate synthetase.

The cloned carAB operon of Escherichia coli coding for the small and large subunits of carbamyl phosphate synthetase has been used to construct a recombinant plasmid with a 4.16 kilobase ClaI fragment of the car operon that lacks the major promoters, P1 and P2. The plasmid, pHN12, carries a functional carB gene. A mutant E. coli strain lacking both subunits of carbamyl phosphate synthetase when transformed with pHN12 overproduces the large subunit by 200-fold (8-10% of the cellular protein). The elevated levels of the large subunit enable the transformed cells to utilize NH3 but not glutamine as nitrogen donor for carbamyl phosphate synthesis. The large subunit has been purified from the overexpressing strain. The purified native large subunit is capable of synthesizing carbamyl phosphate from ammonia, HCO-3, and ATP. The kinetic properties of the large subunit compared with the holoenzyme indicate that the Michaelis constants of the large subunit for HCO-3 and ATP are modulated by its association with the small glutamine binding subunit.

Characterization of a putative promoter region of the human poly(ADP-ribose) polymerase gene: structural similarity to that of the DNA polymerase beta gene.

The 5'-flanking region of the human poly(ADP-ribose) polymerase gene was isolated and characterized. The nucleotide sequence of a part of the poly(ADP-ribose) polymerase gene completely matched that of the cDNA. The transcriptional initiation sites (cap sites) of this gene, located about 166-bp upstream from the translational initiation site, were identified by S1 mapping analysis. Neither CAAT box nor TATA box was found within 500-bp upstream from the cap sites of poly(ADP-ribose) polymerase gene. The 200-bp immediately upstream of the cap site had a high G+C content (76.5%) and contained double repeats of the sequence CCGCCC, putative Sp1 binding sites, and a palindromic structure. The 5'-flanking region of poly(ADP-ribose) polymerase gene also showed promoter activity in chloramphenicol acetyltransferase assay and structural similarity to that of DNA polymerase beta gene.

Yeast carbamyl phosphate synthetase. Structure of the yeast gene and homology to Escherichia coli carbamyl phosphate synthetase.

A cloned fragment of yeast chromosomal DNA carrying the gene CPA2 coding for the large subunit of arginine-specific carbamyl phosphate synthetase has been sequenced. The cloned DNA has a 3,354-nucleotide long continuous reading frame coding for a polypeptide of 1,117 amino acids. The calculated molecular weight of the encoded polypeptide is 123,787, in good agreement with the reported molecular weight of the yeast carbamyl phosphate synthetase large subunit. The amino acid sequence of yeast carbamyl phosphate synthetase is homologous to the recently determined sequence of Escherichia coli carbamyl phosphate synthetase (Nyunoya, H., and Lusty, C. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4629-4633) over almost the entire length of the protein. Like the E. coli large subunit, the yeast enzyme exhibits an extensive internal homology between its NH2- and carboxyl-terminal halves. The internal homology in both the yeast and E. coli proteins indicates that the gene coding for the large subunit of carbamyl phosphate synthetase was derived from a tandem duplication which occurred prior to the divergence of eukaryotes and prokaryotes.