Putative Zinc Finger Motif Research Articles

Site-directed mutations in the potyvirus HC-PRO gene affect helper component activity, virus accumulation, and symptom expression in infected tobacco plants

Helper component (HC-Pro) is a virus-encoded nonstructural protein required for aphid transmission of potyviruses. In the tobacco vein mottling virus (TVMV) polyprotein, HC-Pro represents a 457 residue polypeptide from amino acid position 257 to 713. Previous sequence comparison studies have suggested that mutations of one or two specific amino acid residues in the HC-Pro protein might result in loss of aphid transmission activity. To test this hypothesis, the initial targets were the residues corresponding to these specific amino acids, a lys to glu change and an ile to val change at amino acid positions 307 and 482, respectively, of the TVMV polyprotein, as well as the combination of the two. Two additional mutations within the HC-Pro representing dipeptide changes thr-ser to ile-asp and thr-ala to leu-glu at amino acid positions ( 283 284 ) and ( 368 369 ), respectively, were also tested to further define the effects of mutations in this region on helper component activity. The mutations at positions 482 and ( 368 369 ) had no effect on aphid transmission activity, while mutation at position 307 completely abolished the activity. Except for the 482 mutation, all the mutations also affected symptomatology and virus accumulation in infected plants. Due to the very low concentrations of HC-Pro in plants infected with the ( 283 284 ) mutant, the effect of this dipeptide change on aphid transmission activity could not be assessed. The majority of the tested mutations fall within a putative zinc-finger motif postulated in the cysteine-rich N-terminus of HC-Pro. The possible role of this motif in the potyviruses is further discussed in the light of our present results with TVMV.

Nucleotide sequence and gene organization of the 3'-terminal region of chrysanthemum virus B genomic RNA

DNA clones complementary to the 3'-terminal 3426 nucleotides of the genomic RNA of the carlavirus chrysanthemum virus B (CVB) have been sequenced. The sequence contains six open reading frames (ORFs) which encode putative proteins (in the 5'----3' direction) of Mr 25,749 (ORF2), Mr 11,435 (ORF3), Mr 6984 (ORF4), the triple gene block proteins, a protein Mr 34,638 (ORF5); the coat protein, and a protein of Mr 12,609 (ORF6). The latter protein is basic and contains a putative zinc finger motif. The 5'-proximal ORF1 encodes a product with substantial homology to the C-terminal portions of the putative RNA replicases of two other carlaviruses, potato viruses M and S. The analysis of the minus-sense sequence shows one ORF which encodes a polypeptide of Mr 16,817. The sequenced portion of the CVB genomic RNA contains three short internal non-coding regions, two of which are typical for carlaviruses (those between ORFs 1 and 2, and between ORFs 4 and 5), and one (between ORFs 5 and 6) is unusual. There is significant similarity in the amino acid sequences of the CVB RNA-encoded proteins and the corresponding proteins of other carlaviruses.

Sequence similarity between the mammalian bmi-1 proto-oncogene and the Drosophila regulatory genes Psc and Su(z)2

The bmi-1 proto-oncogene can be activated by Moloney murine leukaemia proviral insertions in E mu-myc transgenic mice. It encodes a highly conserved nuclear protein of 324 amino acids which belongs to a family of proteins containing a putative new zinc-finger. Another closely related member of this family is the mouse protein Mel-18. Here we report on the cloning and characterization of a homologous gene (D-bmi) from Drosophila melanogaster. Our analysis indicates that distinct domains of the mouse Bmi-1 protein, including the putative zinc-finger motif, are highly conserved within the much larger D-Bmi protein. Chromosomal localization and sequence comparison reveal that D-bmi is identical to Posterior Sex Combs (Psc) and indicate that the conserved domains between mouse bmi and Psc are also conserved within Suppressor-2 of Zeste (Su(z)2).

Molecular cloning of matrin F/G: A DNA binding protein of the nuclear matrix that contains putative zinc finger motifs.

We have isolated a 2.7-kilobase rat liver cDNA clone that contains the entire 544-amino acid coding sequence for matrin F/G. This protein has previously been localized to the internal, fibrogranular areas of the nuclear matrix and shown to bind to DNA on nitrocellulose blots. The predicted amino acid sequence from the coding region of this cDNA shows that this protein contains approximately 50% hydrophobic amino acids with secondary structure predictions suggesting a large percentage of beta-sheet regions. No significant homologies were found with any other known proteins, including the nuclear lamins. The predicted amino acid sequence was also searched for DNA binding motifs. Two putative zinc finger motifs were found. In addition, a 7-mer palindromic sequence (Ser-Ser-Thr-Asn-Thr-Ser-Ser) was discovered within one of these zinc finger DNA binding regions. A possible regulatory role for this element is discussed.

Identification of cooperating oncogenes in Eμ-myc transgenic mice by provirus tagging

Mo-MLV infection of Eμ-myc transgenic mice results in a dramatic acceleration of pre-B cell lymphomagenesis. We have used provirus tagging to identify genes that cooperate with the Eμ-myc transgene in B cell transformation. Here we report on the identification of four loci, pim-1, bmi-1, pal-1, and bla-1, which are occupied by proviruses in 35%, 35%, 28%, and 14% of the tumors, respectively. bmi-1, pal-1, and bla-1 represent novel common proviral insertion sites. The bmi-1 gene encodes a 324 amino acid protein with a predominantly nuclear localization. bmi-1 is highly conserved in evolution and contains several motifs frequently found in transcriptional regulators, including a new putative zinc finger motif. No genes have yet been assigned to pal-1 and bla-1. The distribution of proviruses over the four common insertion sites suggests that provirus tagging can be used not only to identify the cooperating oncogenes but also to assign these genes to distinct complementation groups in tumorigenesis.

Refolding and purification of human papillomavirus type 16 E7-IacZ fusion protein expressed in Escherichia coli

Human papillomavirus (HPV) type 16 E7-lacZ fusion protein was produced in Escherichia coli, extracted as inclusion bodies, refolded with reducing reagents, and subjected to gel filtration. The refolded protein was purified by ion-exchange column chromatography, resulting in a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 1H nuclear magnetic resonance spectral changes were observed in the high field methyl region in the presence of Zn 2+ ion, suggesting that the refolded form of the fusion protein is possibly renaturated into the putative zinc finger motif ( C. Edmond and K. H. Vousden, 1989, J. Virol. 63, 2650–2656) and supporting the data of J. A. Rawls, R. Pusztai, and M. Green (1990, J. Virol. 64, 6121–6129) on zinc binding to E7 protein using radioisotopically labeled zinc ion.

Nucleotide sequence and DNA recognition elements of alc, the structural gene which encodes allantoicase, a purine catabolic enzyme of Neurospora crassa

The nitrogen regulatory circuit of Neurospora crassa contains structural genes that encode nitrogen catabolic enzymes which are subject to complex genetic and metabolic regulation. This set of genes is controlled by nitrogen limitation, by specific induction, and by the action of nit-2, a major positive-acting regulatory gene, and nmr, a negative-acting control gene. The complete nucleotide sequence of alc, the gene that encodes allantoicase, a purine catabolic enzyme, is presented. The alc gene contains a single intron, is transcribed from two initiation sites situated approximately 50 nb upstream of the translation start site, and encodes a protein comprised of 354 amino acids. Mobility shift and DNA footprint experiments identified a single binding site for the NIT2 regulatory protein in the alc promoter region. The binding site contains a 10 nucleotide base pair symmetrical sequence which is flanked by two possible core binding sequences, TATCT and TATCG. Mutant NIT2/beta-gal fusion proteins with amino acid substitutions in a putative zinc-finger motif were shown to be completely deficient in the ability to bind to the alc promoter DNA fragment.

An Extended Ubiquitin of Dictyostelium Is Located in the Small Ribosomal Subunit

According to its cDNA sequence, the product of the DUB1 gene of Dictyostelium discoideum, called ubex52, consists of a ubiquitin monomer with a basic COOH-terminal tail of 52 amino acids that includes a putative zinc finger motif. Antipeptide antibodies raised against the COOH-terminal end of the tail indicated that the ubex52 protein is present in all developmental stages of D. discoideum and that similar proteins with apparent molecular masses of 15 to 17 kDa are found in yeast, wheat germ, Drosophila, and mammals. Subcellular fractionation showed that the D. discoideum and Saccharomyces cerevisiae proteins recognized by the antibodies are associated with the ribosomal fraction. After separation and purification of the 40 and 60 S ribosomal subunits of D. discoideum, the ubex52 protein was exclusively recovered in the small subunit.

Poly(ADP-ribose)polymerase: a novel finger protein

By Energy Dispersive X-ray fluorescence we have determined that calf thymus poly(ADP-ribose) polymerase binds two zinc ions per enzyme molecule. Using 65Zn (II) for detection of zinc binding proteins and polypeptides on western blots, we found that the zinc binding sites are localized in a 29 kd N-terminal fragment which is included in the DNA binding domain. Metal depletion and restoration experiments proved that zinc is essential for the binding of this fragment to DNA as tested by Southwestern assay. These results correlate with the existence of two putative zinc finger motifs present in the N-terminal part of the human enzyme. Poly(ADP-ribose)polymerase fingers could be involved in the recognition of DNA strand breaks and therefore in enzyme activation.