Cloning and sequencing of the hyaluronate lyase gene fromPropionibacterium acnes

1997 ◽  
Vol 43 (4) ◽  
pp. 315-321 ◽  
Author(s):  
Bret Steiner ◽  
Sandra Romero-Steiner ◽  
Donna Cruce ◽  
Robert George
Keyword(s):  
Amino Acid ◽  
Propionibacterium Acnes ◽  
Binding Domain ◽  
Leader Sequence ◽  
Cloning And Sequencing ◽  
E Coli ◽  
Amino Terminal ◽  
Hyaluronate Lyase ◽  
Membrane Spanning ◽  
And Staphylococcus Aureus

The hyaluronate lyase (hyaluronidase) gene from Propionibacterium acnes was cloned and sequenced. The gene was isolated on an EcoRI-generated 3-kb piece of DNA. Expression was less in Escherichia coli than in P. acnes; in E. coli, active enzyme was only cell associated and not secreted. The gene is 2256-bp long and codes for a protein of 82 kDa. Amino terminal sequencing of the protein of the partially purified gene indicated the presence of a 32-amino-acid leader sequence. The leader sequence showed a membrane-spanning domain and all of the features usually associated with the leader for a secreted protein. The amino acid sequence is predicted to share homology with the hyaluronidase enzymes from Streptococcus pneumoniae, Streptococcus agalactiae, and Staphylococcus aureus. A potential hyaluronate-binding domain was identified and antibody against this domain was inhibitory to the enzyme.Key words: Propionibacterium acnes, hyaluronidase, leader sequence, hyaluronate binding domain, sequence homology among hyaluronidases.

scholarly journals A specific and unusual nuclear localization signal in the DNA binding domain of the Rev-erb orphan receptors

2003 ◽  
Vol 30 (2) ◽  
pp. 197-211 ◽  
Author(s):  
S Chopin-Delannoy ◽  
S Thenot ◽  
F Delaunay ◽  
E Buisine ◽  
A Begue ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Fusion Protein ◽  
Nuclear Receptors ◽  
Nuclear Localization ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Protein Fusion ◽  
Orphan Receptors ◽  
Amino Terminal

The orphan receptors Rev-erbalpha and Rev-erbbeta are members of the nuclear receptors superfamily and act as transcriptional repressors. Rev-erbalpha is expressed with a robust circadian rhythm and is involved in liver metabolism through repression of the ApoA1 gene, but no role has been yet defined for Rev-erbbeta. To gain better understanding of their function and mode of action, we characterized the proteins encoded by these two genes. Both Rev-erbalpha and Rev-erbbeta proteins were nuclear when transiently transfected in COS-1 cells. The major nuclear location signal (NLS) of Rev-erbalpha is in the amino-terminal region of the protein. Fusion of green fluorescent protein (GFP) to the amino terminus of Rev-erbalpha deletion mutants showed that the NLS is located within a 53 amino acid segment of the DNA binding domain (DBD). The homologous region of Rev-erbbeta fused to GFP also targeted the fusion protein to the nucleus, suggesting that the location of this NLS is conserved among all the Rev-erb group members. Interestingly, members of the phylogenetically closest nuclear orphan receptor group (ROR), which exhibit 58% amino acid identity with Rev-erb in the DBD, do not have their NLS located within the DBD. GFP/DBD. RORalpha or GFP/DBD.RORbeta remained cytoplasmic, in contrast to GFP/DBD. Rev-erb fusion proteins. Alignment of human Rev-erb and ROR DBD amino acid sequences predicted that the two basic residues, K167 and R168, located just upstream from the second zinc finger, could play a critical part in the nuclear localization of Rev-erb proteins. Substitution of these two residues with those found in ROR, in the GFP/DBD. Rev-erb context, resulted in cytoplasmic proteins. In contrast, the reverse mutation of the GFP/DBD. RORalpha towards the Rev-erbalpha residues targeted the fusion protein to the nucleus. Our data demonstrate that Rev-erb proteins contain a functional NLS in the DBD. Its location is unusual within the nuclear receptor superfamily and suggests that Rev-erb orphan receptors control their intracellular localization via a mechanism different from that of other nuclear receptors.

scholarly journals Identification and characterization of PF4varl, a human gene variant of platelet factor 4.

1989 ◽  
Vol 9 (4) ◽  
pp. 1445-1451 ◽  
Author(s):  
C J Green ◽  
R S Charles ◽  
B F Edwards ◽  
P H Johnson
Keyword(s):  
Amino Acid ◽  
Terminal Region ◽  
Leader Sequence ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Coding Sequences ◽  
Amino Terminal ◽  
Arginine Residues ◽  
Carboxy Terminal

A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.

scholarly journals Plasmid-Encoded asp Operon Confers a Proton Motive Metabolic Cycle Catalyzed by an Aspartate-Alanine Exchange Reaction

2002 ◽  
Vol 184 (11) ◽  
pp. 2906-2913 ◽  
Author(s):  
Keietsu Abe ◽  
Fumito Ohnishi ◽  
Kyoko Yagi ◽  
Tasuku Nakajima ◽  
Takeshi Higuchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Expression Vector ◽  
Alcaligenes Faecalis ◽  
E Coli ◽  
Cell Extracts ◽  
Membrane Spanning ◽  
Metabolic Cycle

ABSTRACT Tetragenococcus halophila D10 catalyzes the decarboxylation of l-aspartate with nearly stoichiometric release of l-alanine and CO2. This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an l-aspartate-β-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter → aspD → aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known l-aspartate-β-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of l-aspartate-β-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.

scholarly journals nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain.

1991 ◽  
Vol 11 (11) ◽  
pp. 5735-5745 ◽  
Author(s):  
G F Yuan ◽  
Y H Fu ◽  
G A Marzluf
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Neurospora Crassa ◽  
Transcriptional Activation ◽  
Regulatory Gene ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Dna Binding Domain ◽  
Amino Terminal ◽  
Rich Domain

nit-4, a pathway-specific regulatory gene in the nitrogen circuit of Neurospora crassa, is required for the expression of nit-3 and nit-6, the structural genes which encode nitrate and nitrite reductase, respectively. The complete nucleotide sequence of the nit-4 gene has been determined. The predicted NIT4 protein contains 1,090 amino acids and appears to possess a single Zn(II)2Cys6 binuclear-type zinc finger, which may mediate DNA binding. Site-directed mutagenesis studies demonstrated that cysteine and other conserved amino acid residues in this possible DNA-binding domain are necessary for nit-4 function. A stretch of 27 glutamines, encoded by a CAGCAA repeating sequence, occurs in the C terminus of the NIT4 protein, and a second glutamine-rich domain occurs further upstream. A NIT4 protein deleted for the polyglutamine region was still functional in vivo. However, nit-4 function was abolished when both the polyglutamine region and the glutamine-rich domain were deleted, suggesting that the glutamine-rich domain might function in transcriptional activation. The homologous regulatory gene from Aspergillus nidulans, nirA, encodes a protein whose amino-terminal half has approximately 60% amino acid identity with NIT4 but whose carboxy terminus is completely different. A hybrid nit-4-nirA gene was constructed and found to function in N. crassa.

Synthesis of an enzymatically active FLP recombinase in vitro: search for a DNA-binding domain

1989 ◽  
Vol 9 (5) ◽  
pp. 1987-1995
Author(s):  
A A Amin ◽  
P D Sadowski
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Binding Domain ◽  
Translation System ◽  
Dna Binding Domain ◽  
Amino Terminal ◽  
Dna Binding Activity

We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein. The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP. Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination. Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity. Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity. On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity. Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95%. In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination. On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain. The possible reasons for this result are discussed.

scholarly journals Sequence of Events Underlying the Allosteric Transition of Rod Cyclic Nucleotide–gated Channels

1999 ◽  
Vol 113 (5) ◽  
pp. 621-640 ◽  
Author(s):  
Elizabeth R. Sunderman ◽  
William N. Zagotta
Keyword(s):  
Amino Acid ◽  
Cyclic Nucleotide ◽  
Binding Domain ◽  
Terminal Region ◽  
Allosteric Transition ◽  
Amino Terminal ◽  
Sequence Of Events ◽  
Carboxyl Terminal ◽  
Purine Ring ◽  
Closing Rate

Activation of cyclic nucleotide–gated (CNG) ion channels involves a conformational change in the channel protein referred to as the allosteric transition. The amino terminal region and the carboxyl terminal cyclic nucleotide–binding domain of CNG channels have been shown to be involved in the allosteric transition, but the sequence of molecular events occurring during the allosteric transition is unknown. We recorded single-channel currents from bovine rod CNG channels in which mutations had been introduced in the binding domain at position 604 and/or the rat olfactory CNG channel amino terminal region had been substituted for the bovine rod amino terminal region. Using a hidden Markov modeling approach, we analyzed the kinetics of these channels activated by saturating concentrations of cGMP, cIMP, and cAMP. We used thermodynamic mutant cycles to reveal an interaction during the allosteric transition between the purine ring of the cyclic nucleotides and the amino acid at position 604 in the binding site. We found that mutations at position 604 in the binding domain alter both the opening and closing rate constants for the allosteric transition, indicating that the interactions between the cyclic nucleotide and this amino acid are partially formed at the time of the transition state. In contrast, the amino terminal region affects primarily the closing rate constant for the allosteric transition, suggesting that the state-dependent stabilizing interactions between amino and carboxyl terminal regions are not formed at the time of the transition state for the allosteric transition. We propose that the sequence of events that occurs during the allosteric transition involves the formation of stabilizing interactions between the purine ring of the cyclic nucleotide and the amino acid at position 604 in the binding domain followed by the formation of stabilizing interdomain interactions.

scholarly journals Cloning and sequencing of a novel gene (recG) that affects the quinolone susceptibility of Staphylococcus aureus.

1997 ◽  
Vol 41 (8) ◽  
pp. 1770-1774 ◽  
Author(s):  
T Niga ◽  
H Yoshida ◽  
H Hattori ◽  
S Nakamura ◽  
H Ito
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Gene Product ◽  
Parent Strain ◽  
Amino Acid Identity ◽  
Sequencing Analysis ◽  
Deficient Mutant ◽  
Cloning And Sequencing ◽  
Acid Identity ◽  
E Coli

In a study of the quinolone resistance genes in Staphylococcus aureus, a recG homolog was cloned as a gene affecting quinolone susceptibility. Sequencing analysis revealed that the gene consists of 2,061 nucleotides and encodes a 686-amino-acid polypeptide, which shows 38, 39, and 50% amino acid identity with the RecGs of Escherichia coli, Haemophilus influenzae, and Streptococcus pneumoniae, respectively. Seven helicase motifs are well conserved in the gene product. A plasmid carrying the gene complemented a recG-deficient mutant of E. coli with respect to mitomycin hypersusceptibility, demonstrating that the gene product is functionally equivalent to E. coli RecG. These results indicate that the gene is the recG gene of S. aureus. S. aureus RCM101 (recG::Tn551), designated S. aureus 3f33, is four to eight times more susceptible to quinolones than the parent strain, RCM101. The transformation of strain 3f33 with a plasmid carrying the S. aureus recG gene made it as quinolone resistant as strain RCM101. These results suggest that the recG gene is involved in the repair of DNA damage resulting from quinolone treatment in S. aureus.

Identification and characterization of PF4varl, a human gene variant of platelet factor 4

1989 ◽  
Vol 9 (4) ◽  
pp. 1445-1451
Author(s):  
C J Green ◽  
R S Charles ◽  
B F Edwards ◽  
P H Johnson
Keyword(s):  
Amino Acid ◽  
Terminal Region ◽  
Leader Sequence ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Coding Sequences ◽  
Amino Terminal ◽  
Arginine Residues ◽  
Carboxy Terminal

A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.

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