Poly(ADP-ribosyl)ation of p53 In Vitro and In Vivo Modulates Binding to its DNA Consensus Sequence

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

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Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems.

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.5073 ◽

1989 ◽

Vol 9 (11) ◽

pp. 5073-5080 ◽

Cited By ~ 314

Author(s):

M Kozak

Keyword(s):

Context Effects ◽

Consensus Sequence ◽

Negative Regulator ◽

Late Event ◽

Free Translation ◽

Initiator Codon ◽

Translation Systems ◽

Short Versions

The context requirements for recognition of an initiator codon were evaluated in vitro by monitoring the relative use of two AUG codons that were strategically positioned to produce long (pre-chloramphenicol acetyl transferase [CAT]) and short versions of CAT protein. The yield of pre-CAT initiated from the 5'-proximal AUG codon increased, and synthesis of CAT from the second AUG codon decreased, as sequences flanking the first AUG codon increasingly resembled the eucaryotic consensus sequence. Thus, under prescribed conditions, the fidelity of initiation in extracts from animal as well as plant cells closely mimics what has been observed in vivo. Unexpectedly, recognition of an AUG codon in a suboptimal context was higher when the adjacent downstream sequence was capable of assuming a hairpin structure than when the downstream region was unstructured. This finding adds a new, positive dimension to regulation by mRNA secondary structure, which has been recognized previously as a negative regulator of initiation. Translation of pre-CAT from an AUG codon in a weak context was not preferentially inhibited under conditions of mRNA competition. That result is consistent with the scanning model, which predicts that recognition of the AUG codon is a late event that occurs after the competition-sensitive binding of a 40S ribosome-factor complex to the 5' end of mRNA. Initiation at non-AUG codons was evaluated in vitro and in vivo by introducing appropriate mutations in the CAT and preproinsulin genes. GUG was the most efficient of the six alternative initiator codons tested, but GUG in the optimal context for initiation functioned only 3 to 5% as efficiently as AUG. Initiation at non-AUG codons was artifactually enhanced in vitro at supraoptimal concentrations of magnesium.

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Repeated CT elements bound by zinc finger proteins control the absolute and relative activities of the two principal human c-myc promoters

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5710-5724.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5710-5724

Author(s):

E DesJardins ◽

N Hay

Keyword(s):

Zinc Finger ◽

Tandem Repeats ◽

Zinc Finger Protein ◽

Consensus Sequence ◽

Regulatory Region ◽

Maximal Activity ◽

Single Copy ◽

Promoter Usage

Transcription of the human proto-oncogene c-myc is governed by two tandem principal promoters, termed P1 and P2. In general, the downstream promoter, P2, is predominant, which is in contrast to the promoter occlusion phenomenon usually observed in genes containing tandem promoters. A shift in human c-myc promoter usage has been observed in some tumor cells and in certain physiological conditions. However, the mechanisms that regulate promoter usage are not well understood. The present studies identify regulators which are required to promote transcription from both human c-myc promoters, P1 and P2, and have a role in determining their relative activities in vivo. A novel regulatory region located 101 bp upstream of P1 was characterized and contains five tandem repeats of the consensus sequence CCCTCCCC (CT element). The integrity of the region containing all five elements is required to promote transcription from P1 and for maximal activity from P2 in vivo. A single copy of this same element, designated CT-I2, also appears in an inverted orientation 53 bp upstream of the P2 transcription start site. This element has an inhibitory effect on P1 transcription and is required for P2 transcription. The transcription factor Sp1 was identified as the factor that binds specifically to the tandem CT elements upstream of P1 and to the CT-I2 element upstream of P2. In addition, the recently cloned zinc finger protein ZF87, or MAZ, was also able to bind these same elements in vitro. The five tandem CT elements can be functionally replaced by a heterologous enhancer that only in the absence of CT-I2 reverses the promoter usage, similar to what is observed in the translocated c-myc allele of Burkitt's lymphoma cells.

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ParB of Pseudomonas aeruginosa: Interactions with Its Partner ParA and Its Target parS and Specific Effects on Bacterial Growth

Journal of Bacteriology ◽

10.1128/jb.186.20.6983-6998.2004 ◽

2004 ◽

Vol 186 (20) ◽

pp. 6983-6998 ◽

Cited By ~ 68

Author(s):

Aneta A. Bartosik ◽

Krzysztof Lasocki ◽

Jolanta Mierzejewska ◽

Christopher M. Thomas ◽

Grazyna Jagura-Burdzy

Keyword(s):

Pseudomonas Aeruginosa ◽

Streptomyces Coelicolor ◽

Consensus Sequence ◽

Terminal Part ◽

Dimerization Domain ◽

Interaction Domain ◽

C Terminus ◽

Cell Components

ABSTRACT The par genes of Pseudomonas aeruginosa have been studied to increase the understanding of their mechanism of action and role in the bacterial cell. Key properties of the ParB protein have been identified and are associated with different parts of the protein. The ParB- ParB interaction domain was mapped in vivo and in vitro to the C-terminal 56 amino acids (aa); 7 aa at the C terminus play an important role. The dimerization domain of P. aeruginosa ParB is interchangeable with the dimerization domain of KorB from plasmid RK2 (IncP1 group). The C-terminal part of ParB is also involved in ParB-ParA interactions. Purified ParB binds specifically to DNA containing a putative parS sequence based on the consensus sequence found in the chromosomes of Bacillus subtilis, Pseudomonas putida, and Streptomyces coelicolor. The overproduction of ParB was shown to inhibit the function of genes placed near parS. This “silencing” was dependent on the parS sequence and its orientation. The overproduction of P. aeruginosa ParB or its N-terminal part also causes inhibition of the growth of P. aeruginosa and P. putida but not Escherichia coli cells. Since this inhibitory determinant is located well away from ParB segments required for dimerization or interaction with the ParA counterpart, this result may suggest a role for the N terminus of P. aeruginosa ParB in interactions with host cell components.

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Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helix-loop-helix proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4478 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4478-4485 ◽

Cited By ~ 68

Author(s):

L Li ◽

R Heller-Harrison ◽

M Czech ◽

E N Olson

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Transcriptional Activation ◽

Consensus Sequence ◽

Signal Transduction Pathway ◽

Specific Gene ◽

Dependent Protein Kinase ◽

Dependent Protein

Differentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-specific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin, myf5, and MRF4. Repression by the PKA catalytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators. Phosphopeptide mapping of myogenin in vitro and in vivo revealed two PKA phosphorylation sites, both within the basic region. However, repression of myogenin function by PKA does not require direct phosphorylation of these sites but instead involves an indirect mechanism with one or more intermediate steps. Regulation of the transcriptional activity of the MyoD family by modulation of the cAMP signaling pathway may account for the inhibitory effects of certain peptide growth factors on muscle-specific gene expression and may also determine the responsiveness of different cell types to myogenic conversion by these myogenic regulators.

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Identification of a Novel Virulence Factor in Recombinant Pneumonia Virus of Mice

Journal of Virology ◽

10.1128/jvi.00364-07 ◽

2007 ◽

Vol 81 (17) ◽

pp. 9490-9501 ◽

Cited By ~ 18

Author(s):

Christine D. Krempl ◽

Anna Wnekowicz ◽

Elaine W. Lamirande ◽

Giw Nayebagha ◽

Peter L. Collins ◽

...

Keyword(s):

Virulence Factor ◽

Reverse Genetics ◽

Fluorescent Protein ◽

Vaccine Development ◽

Virulent Strain ◽

Consensus Sequence ◽

Cytoplasmic Tail ◽

Syncytial Virus

ABSTRACT Pneumonia virus of mice (PVM) is a murine relative of human respiratory syncytial virus (HRSV). Here we developed a reverse genetics system for PVM based on a consensus sequence for virulent strain 15. Recombinant PVM and a version engineered to express green fluorescent protein replicated as efficiently as the biological parent in vitro but were 4- and 12.5-fold attenuated in vivo, respectively. The G proteins of HRSV and PVM have been suggested to contribute to viral pathogenesis, but this had not been possible to study in a defined manner in a fully permissive host. As a first step, we evaluated recombinant mutants bearing a deletion of the entire G gene (ΔG) or expressing a G protein lacking its cytoplasmic tail (Gt). Both G mutants replicated as efficiently in vitro as their recombinant parent, but both were nonpathogenic in mice at doses that would otherwise be lethal. We could not detect replication of the ΔG mutant in mice, indicating that its attenuation is based on a severe reduction in the virus load. In contrast, the Gt mutant appeared to replicate as efficiently in mice as its recombinant parent. Thus, the reduction in virulence associated with the Gt mutant could not be accounted for by a reduction in viral replication. These results identified the cytoplasmic tail of G as a virulence factor whose effect is not mediated solely by the viral load. In addition to its intrinsic interest, a recombinant virus that replicates with wild-type-like efficiency but does not cause disease defines optimal properties for vaccine development.

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N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4456-4465.1990 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4456-4465

Author(s):

S M Carroll ◽

P Narayan ◽

F M Rottman

Keyword(s):

Consensus Sequence ◽

Specific Sequence ◽

Free System ◽

Consensus Sequences ◽

Neplanocin A ◽

A Cell ◽

Steady State Levels ◽

Precursor Rna

N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.

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Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helix-loop-helix proteins

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4478-4485.1992 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4478-4485

Author(s):

L Li ◽

R Heller-Harrison ◽

M Czech ◽

E N Olson

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Transcriptional Activation ◽

Consensus Sequence ◽

Signal Transduction Pathway ◽

Specific Gene ◽

Dependent Protein Kinase ◽

Dependent Protein

Differentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-specific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin, myf5, and MRF4. Repression by the PKA catalytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators. Phosphopeptide mapping of myogenin in vitro and in vivo revealed two PKA phosphorylation sites, both within the basic region. However, repression of myogenin function by PKA does not require direct phosphorylation of these sites but instead involves an indirect mechanism with one or more intermediate steps. Regulation of the transcriptional activity of the MyoD family by modulation of the cAMP signaling pathway may account for the inhibitory effects of certain peptide growth factors on muscle-specific gene expression and may also determine the responsiveness of different cell types to myogenic conversion by these myogenic regulators.

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Participation of the yeast activator Abf1 in meiosis-specific expression of the HOP1 gene.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.2777 ◽

1996 ◽

Vol 16 (6) ◽

pp. 2777-2786 ◽

Cited By ~ 24

Author(s):

V Gailus-Durner ◽

J Xie ◽

C Chintamaneni ◽

A K Vershon

Keyword(s):

Transcriptional Activation ◽

Mutational Analysis ◽

Consensus Sequence ◽

Promoter Sequence ◽

Regulatory Elements ◽

Specific Gene ◽

Specific Expression ◽

Autonomously Replicating Sequence

The meiosis-specific gene HOP1, which encodes a component of the synaptonemal complex, is controlled through two regulatory elements, UASH and URS1H. Sites similar to URS1H have been identified in the promoter region of virtually every early meiosis-specific gene, as well as in many promoters of nonmeiotic genes, and it has been shown that the proteins that bind to this site function to regulate meiotic and nonmeiotic transcription. Sites similar to the UASH site have been found in a number of meiotic and nonmeiotic genes as well. Since it has been shown that UASH functions as an activator site in vegetative haploid cells, it seemed likely that the factors binding to this site regulate both meiotic and nonmeiotic transcription. We purified the factor binding to the UASH element of the HOP1 promoter. Sequence analysis identified the protein as Abf1 (autonomously replicating sequence-binding factor 1), a multifunctional protein involved in DNA replication, silencing, and transcriptional regulation. We show by mutational analysis of the UASH site, that positions outside of the proposed UASH consensus sequence (TNTGN[A/T]GT) are required for DNA binding in vitro and transcriptional activation in vivo. A new UASH consensus sequence derived from this mutational analysis closely matches a consensus Abf1 binding site. We also show that an Abf1 site from a nonmeiotic gene can replace the function of the UASH site in the HOP1 promoter. Taken together, these results show that Abf1 functions to regulate meiotic gene expression.

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Sequence of the Spacer in the Recombination Signal Sequence Affects V(D)J Rearrangement Frequency and Correlates with Nonrandom Vκ Usage In Vivo

Journal of Experimental Medicine ◽

10.1084/jem.187.9.1495 ◽

1998 ◽

Vol 187 (9) ◽

pp. 1495-1503 ◽

Cited By ~ 55

Author(s):

Bertrand Nadel ◽

Alan Tang ◽

Guia Escuro ◽

Geanncarlo Lugo ◽

Ann J. Feeney

Keyword(s):

Relative Frequency ◽

Signal Sequence ◽

Consensus Sequence ◽

Recombination Signal Sequence ◽

Recombination Rates ◽

Recombination Signal ◽

Functional Variable ◽

Determinant Factor

Functional variable (V), diversity (D), and joining (J) gene segments contribute unequally to the primary repertoire. One factor contributing to this nonrandom usage is the relative frequency with which the different gene segments rearrange. Variation from the consensus sequence in the heptamer and nonamer of the recombination signal sequence (RSS) is therefore considered a major factor affecting the relative representation of gene segments in the primary repertoire. In this study, we show that the sequence of the spacer is also a determinant factor contributing to the frequency of rearrangement. Moreover, the effect of the spacer on recombination rates of various human Vκ gene segments in vitro correlates with their frequency of rearrangement in vivo in pre-B cells and with their representation in the peripheral repertoire.

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