Design of artificial sequence-specific DNA bending ligands.

Proteins that bend DNA are important regulators of biological processes. Sequence-specific DNA bending ligands have been designed that bind two noncontiguous sites in the major groove and induce a bend in the DNA. An oligonucleotide containing pyrimidine segments separated by a central variable linker domain simultaneously binds by triple helix formation two 15-bp purine tracts separated by 10 bp. Bend angles of 61 degrees, 50 degrees, and 38 degrees directed towards the minor groove were quantitated by phasing analysis for linkers of four, five, and six T residues, respectively. The design and synthesis of nonnatural architectural factors may provide a new class of reagents for use in biology and human medicine.

The melanoma differentiation associated gene mda-7 suppresses cancer cell growth.

Cancer is a disease characterized by defects in growth control, and tumor cells often display abnormal patterns of cellular differentiation. The combination of recombinant human fibroblast interferon and the antileukemic agent mezerein corrects these abnormalities in cultured human melanoma cells resulting in irreversible growth arrest and terminal differentiation. Subtraction hybridization identifies a melanoma differentiation associated gene (mda-7) with elevated expression in growth arrested and terminally differentiated human melanoma cells. Colony formation decreases when mda-7 is transfected into human tumor cells of diverse origin and with multiple genetic defects. In contrast, the effects of mda-7 on growth and colony formation in transient transfection assays with normal cells, including human mammary epithelial, human skin fibroblast, and rat embryo fibroblast, is quantitatively less than that found with cancer cells. Tumor cells expressing elevated mda-7 display suppression in monolayer growth and anchorage independence. Infection with a recombinant type 5 adenovirus expressing antisense mda-7 eliminates mda-7 suppression of the in vitro growth and transformed phenotype. The ability of mda-7 to suppress growth in cancer cells not expressing or containing defects in both the retinoblastoma (RB) and p53 genes indicates a lack of involvement of these critical tumor suppressor elements in mediating mda-7-induced growth inhibition. The lack of protein homology of mda-7 with previously described growth suppressing genes and the differential effect of this gene on normal versus cancer cells suggests that mda-7 may represent a new class of cancer growth suppressing genes with antitumor activity.

Sequence tag identification of intact proteins by matching tanden mass spectral data against sequence data bases.

Molecular and fragment ion data of intact 8- to 43-kDa proteins from electrospray Fourier-transform tandem mass spectrometry are matched against the corresponding data in sequence data bases. Extending the sequence tag concept of Mann and Wilm for matching peptides, a partial amino acid sequence in the unknown is first identified from the mass differences of a series of fragment ions, and the mass position of this sequence is defined from molecular weight and the fragment ion masses. For three studied proteins, a single sequence tag retrieved only the correct protein from the data base; a fourth protein required the input of two sequence tags. However, three of the data base proteins differed by having an extra methionine or by missing an acetyl or heme substitution. The positions of these modifications in the protein examined were greatly restricted by the mass differences of its molecular and fragment ions versus those of the data base. To characterize the primary structure of an unknown represented in the data base, this method is fast and specific and does not require prior enzymatic or chemical degradation.

Binding of a hairpin polyamide in the minor groove of DNA: sequence-specific enthalpic discrimination.

Hairpin polyamides are synthetic ligands for sequence-specific recognition in the minor groove of double-helical DNA. A thermodynamic characterization of the DNA-binding properties exhibited by a six-ring hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (where Im = imidazole, Py = pyrrole, gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide), reveals an approximately 1-2 kcal/mol greater affinity for the designated match site, 5'-TGTTA-3', relative to the single base pair mismatch sites, 5'-TGGTA-3' and 5'-TATTA-3'. The enthalpy and entropy data at 20 degrees C reveal this sequence specificity to be entirely enthalpic in origin. Correlations between the thermodynamic driving forces underlying the sequence specificity exhibited by ImPyPy-gamma-PyPyPy-beta-Dp and the structural properties of the heterodimeric complex of PyPyPy and ImPyPy bound to the minor groove of DNA provide insight into the molecular forces that govern the affinity and specificity of pyrrole-imidazole polyamides.

Megalin-mediated endocytosis of transcobalamin-vitamin-B12 complexes suggests a role of the receptor in vitamin-B12 homeostasis.

Kidney cortex is a main target for circulating vitamin B12 (cobalamin) in complex with transcobalamin (TC). Ligand blotting of rabbit kidney cortex with rabbit 125I-TC-B12 and human TC-57Co-B12 revealed an exclusive binding to megalin, a 600-kDa endocytic receptor present in renal proximal tubule epithelium and other absorptive epithelia. The binding was Ca2+ dependent and inhibited by receptor-associated protein (RAP). Surface plasmon resonance analysis demonstrated a high-affinity interaction between purified rabbit megalin and rabbit TC-B12 but no measurable affinity of the vitamin complex for the homologous alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor related protein (LRP). 125I-TC-B12 was efficiently endocytosed in a RAP-inhibitable manner in megalin-expressing rat yolk sac carcinoma cells and in vivo microperfused rat proximal tubules. The radioactivity in the tubules localized to the endocytic compartments and a similar apical distribution in the proximal tubules was demonstrated after intravenous injection of 125I-TC-B12. The TC-B12 binding sites in the proximal tubule epithelium colocalized with megalin as shown by ligand binding to cryosections of rat kidney cortex, and the binding was inhibited by anti-megalin polyclonal antibody, EDTA, and RAP. These data show a novel nutritional dimension of megalin as a receptor involved in the cellular uptake of vitamin B12. The expression of megalin in absorptive epithelia in the kidney and other tissues including yolk sac and placenta suggests a role of the receptor in vitamin B12 homeostasis and fetal vitamin B12 supply.

Environmental and developmental signals modulate proline homeostasis: evidence for a negative transcriptional regulator.

In many plants, osmotic stress induces a rapid accumulation of proline through de novo synthesis from glutamate. This response is thought to play a pivotal role in osmotic stress tolerance [Kishor, P. B. K., Hong, Z., Miao, G.-H., Hu, C.-A. A. and Verma, D. P. S. (1995) Plant Physiol. 108, 1387-1394]. During recovery from osmotic stress, accumulated proline is rapidly oxidized to glutamate and the first step of this process is catalyzed by proline oxidase. We have isolated a full-length cDNA from Arabidopsis thaliana, At-POX, which maps to a single locus on chromosome 3 and that encodes a predicted polypeptide of 499 amino acids showing significant similarity with proline oxidase sequences from Drosophila and Saccharomyces cerevisiae (55.5% and 45.1%, respectively). The predicted location of the encoded polypeptide is the inner mitochondrial membrane. RNA gel blot analysis revealed that At-POX mRNA levels declined rapidly upon osmotic stress and this decline preceded proline accumulation. On the other hand, At-POX mRNA levels rapidly increased during recovery. Free proline, exogenously added to plants, was found to be an effective inducer of At-POX expression; indeed, At-POX was highly expressed in flowers and mature seeds where the proline level is higher relative to other organs of Arabidopsis. Our results indicate that stress- and developmentally derived signals interact to determine proline homeostasis in Arabidopsis.

Binding site size limit of the 2:1 pyrrole-imidazole polyamide-DNA motif.

Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids can be combined in antiparallel side-by-side dimeric complexes for sequence-specific recognition in the minor groove of DNA. Six polyamides containing three to eight rings bind DNA sites 5-10 bp in length, respectively. Quantitative DNase I footprint titration experiments demonstrate that affinity maximizes and is similar at ring sizes of five, six, and seven. Sequence specificity decreases as the length of the polyamides increases beyond five rings. These results provide useful guidelines for the design of new polyamides that bind longer DNA sites with enhanced affinity and specificity.

Surface-epitope masking and expression cloning identifies the human prostate carcinoma tumor antigen gene PCTA-1 a member of the galectin gene family.

The selective production of monoclonal antibodies (mAbs) reacting with defined cell surface-expressed molecules is now readily accomplished with an immunological subtraction approach, surface-epitope masking (SEM). Using SEM, prostate carcinoma (Pro 1.5) mAbs have been developed that react with tumor-associated antigens expressed on human prostate cancer cell lines and patient-derived carcinomas. Screening a human LNCaP prostate cancer cDNA expression library with the Pro 1.5 mAb identifies a gene, prostate carcinoma tumor antigen-1 (PCTA-1). PCTA-1 encodes a secreted protein of approximately 35 kDa that shares approximately 40% sequence homology with the N-amino terminal region of members of the S-type galactose-binding lectin (galectin) gene family. Specific galectins are found on the surface of human and marine neoplastic cells and have been implicated in tumorigenesis and metastasis. Primer pairs within the 3' untranslated region of PCTA-1 and reverse transcription-PCR demonstrate selective expression of PCTA-1 by prostate carcinomas versus normal prostate and benign prostatic hypertrophy. These findings document the use of the SEM procedure for generating mAbs reacting with tumor-associated antigens expressed on human prostate cancers. The SEM-derived mAbs have been used for expression cloning the gene encoding this human tumor antigen. The approaches described in this paper, SEM combined with expression cloning, should prove of wide utility for developing immunological reagents specific for and identifying genes relevant to human cancer.

A gain-of-function of an amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutant: An enhancement of free radical formation due to a decrease in Km for hydrogen peroxide.

Cu,Zn-superoxide dismutase (SOD) is known to be a locus of mutation in familial amyotrophic lateral sclerosis (FALS). Transgenic mice that express a mutant Cu,Zn-SOD, Gly-93--> Ala (G93A), have been shown to develop amyotrophic lateral sclerosis (ALS) symptoms. We cloned the FALS mutant, G93A, and wild-type cDNA of human Cu,Zn-SOD, overexpressed them in Sf9 insect cells, purified the proteins, and studied their enzymic activities for catalyzing the dismutation of superoxide anions and the generation of free radicals with H2O2 as substrate. Our results showed that both enzymes contain one copper ion per subunit and have identical dismutation activity. However, the free radical-generating function of the G93A mutant, as measured by the spin trapping method, is enhanced relative to that of the wild-type enzyme, particularly at lower H2O2 concentrations. This is due to a small, but reproducible, decrease in the value of Km for H2O2 for the G93A mutant, while the kcat is identical for both enzymes. Thus, the ALS symptoms observed in G93A transgenic mice are not caused by the reduction of Cu,Zn-SOD activity with the mutant enzyme; rather, it is induced by a gain-of-function, an enhancement of the free radical-generating function. This is consistent with the x-ray crystallographic studies showing the active channel of the FALS mutant is slightly larger than that of the wild-type enzyme; thus, it is more accessible to H2O2. This gain-of-function, in part, may provide an explanation for the association between ALS and Cu,Zn-SOD mutants.

Identification of functional domains and evolution of Tc1-like transposable elements.

Tc1-like transposable elements from teleost fish have been phylogenetically examined to determine the mechanisms involved in their evolution and conserved domains of function. We identified two new functional domains in these elements. The first is a bipartite nuclear localization signal, indicating that transposons can take advantage of the transport machinery of host cells for nuclear uptake of their transposases. The second is a novel combination of a paired domain-related protein motif juxtaposed to a leucine zipper-like domain located in the putative DNA-binding regions of the transposases. This domain coexists with a special inverted repeat structure in certain transposons in such phylogenetically distant hosts as fish and insects. Our data indicate that reassortment of functional domains and horizontal transmission between species are involved in the formation and spread of new types of transposable elements.

Peroxynitrite disables the tyrosine phosphorylation regulatory mechanism: Lymphocyte-specific tyrosine kinase fails to phosphorylate nitrated cdc2(6-20)NH2 peptide.

To determine if nitration of tyrosine residues by peroxynitrite (PN), which can be generated endogenously, can disrupt the phosphorylation of tyrosine residues in proteins involved in cell signaling networks, we studied the effect of PN-promoted nitration of tyrosine residues in a pentadecameric peptide, cdc2(6-20)NH2, on the ability of the peptide to be phosphorylated. cdc2(6-20)NH2 corresponds to the tyrosine phosphorylation site of p34cdc2 kinase, which is phosphorylated by lck kinase (lymphocyte-specific tyrosine kinase, p56lck). PN nitrates both Tyr-15 and Tyr-19 of the peptide in phosphate buffer (pH 7.5) at 37 degrees C. Nitration of Tyr-15. which is the phosphorylated amino acid residue, inhibits completely the phosphorylation of the peptide. The nitration reaction is enhanced by either Fe(III)EDTA or Cu(II)-Zn(II)-superoxide dismutase (Cu,Zn-SOD). The kinetic data are consistent with the view that reactions of Fe(111)EDTA or Cu,Zn-SOD with the cis form of PN yield complexes in which PN decomposes more slowly to form N02+, the nitrating agent. Thus, the nitration efficiency of PN is enhanced. These results are discussed from the point of view that PN-promoted nitration will result in permanent impairment of cyclic cascades that control signal transduction processes and regulate cell cycles.

Regulation of meiotic chromatin loop size by chromosomal position.

At meiotic prophase, chromatin loops around a proteinaceous core, with the sizes of these loops varying between species. Comparison of the morphology of sequence-related inserts at different sites in transgenic mice demonstrates that loop size also varies with chromosomal geography. Similarly, chromatin loop lengths differ dramatically for interstitially and terminally located hamster telomeric sequences. Sequences, telomeric or otherwise, located at chromosome termini, closely associate with the meiotic proteinaceous core, forming shorter loops than identical interstitial sequences. Thus, we present evidence that different chromatin packaging mechanisms exist for interstitial versus terminal chromosomal regions, which act separately from those operating at the level of the DNA sequence. Chromosomal position plays the dominant role in chromatin packaging.

Peroxynitrite-mediated nitration of tyrosine residues in Escherichia coli glutamine synthetase mimics adenylylation: relevance to signal transduction.

Treatment of Escherichia coli glutamine synthetase (GS) with peroxynitrite leads to nitration of some tyrosine residues and conversion of some methionine residues to methionine sulfoxide (MSOX) residues. Nitration, but not MSOX formation, is stimulated by Fe-EDTA. In the absence of Fe-EDTA, nitration of only one tyrosine residue per subunit of unadenylylated GS leads to changes in divalent cation requirement, pH-activity profile, affinity for ADP, and susceptibility to feedback inhibition by end products (tryptophan, AMP, CTP), whereas nitration of one tyrosine residue per subunit in the adenylylated GS leads to complete loss of catalytic activity. In the presence of Fe-EDTA, nitration is a more random process: nitration of five to six tyrosine residues per subunit is needed to convert unadenylylated GS to the adenylylated configuration. These results and the fact that nitration of tyrosine residues is an irreversible process serve notice that the regulatory function of proteins that undergo phosphorylation or adenylylation in signal transduction cascades might be seriously compromised by peroxynitrite-promoted nitration.

DANA elements: a family of composite, tRNA-derived short interspersed DNA elements associated with mutational activities in zebrafish (Danio rerio).

DNA is the first SINE isolated from zebrafish (Danio rerio) exhibiting all the hallmarks of these tRNA-derived elements. DANA is unique in its clearly defined substructure of distinct cassettes. In contrast to generic SINE elements, DANA appears to have been assembled by insertions of short sequences into a progenitor, tRNA-derived element. Once associated with each other, these subunits were amplified as a new transposable element with such a remarkable success that DANA-related sequences comprise approximately 10% of the modern zebrafish genome. At least some of the sequences comprised by the full-length element were capable of movement, forming a new group of mobile, composite transposons, one of which caused an insertional mutation in the zebrafish no tail gene. Being present only in the genus Danio, and estimated to be as old as the genus itself, DANA may have played a role in Danio speciation by massive amplification and genome-wide dispersion. There are extensive DNA polymorphisms between zebrafish populations and strains detected by PCR amplification using primers specific to DANA, suggesting that the DANA element will be useful as a molecular tool for genetic and phylogenetic analyses.

Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain.

Pleckstrin homology (PH) domains are found in many signaling molecules and are thought to be involved in specific intermolecular interactions. Their binding to several proteins and to membranes containing 1-alpha-phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been reported. A region that includes the PH domain has also been implicated in binding of phospholipase C-delta 1 (PLC-delta 1) to both PtdIns(4,5)P2 and D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] [Cifuentes, M. E., Delaney, T. & Rebecchi, M. J. (1994) J. Biol. Chem. 269, 1945-1948]. We report herein that the isolated PH domain from PLC-delta 1 binds to both PtdIns(4,5)P2 and Ins(1,4,5)P3 with high affinity and shows the same binding specificity seen by others with whole PLC-delta 1. Thus the PH domain is functionally and structurally modular. These results demonstrate stereo-specific high-affinity binding by an isolated PH domain and further support a functional role for PH domains in the regulation of PLC isoforms. Other PH domains did not bind strongly to the compounds tested, suggesting that inositol phosphates and phospholipids are not likely physiological ligands for all PH domains. Nonetheless, since all PH-domain-containing proteins are associated with membrane surfaces, several PH domains bind to specific sites on membranes, and PH domains appear to be electrostatically polarized, a possible general role for PH domains in membrane association is suggested.