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PubMed1995

Structure, function and evolution of seryl-tRNA synthetases: implications for the evolution of aminoacyl-tRNA synthetases and the genetic code.

Two aspects of the evolution of aminoacyl-tRNA synthetases are discussed. Firstly, using recent crystal structure information on seryl-tRNA synthetase and its substrate complexes, the coevolution of the mode of recognition between seryl-tRNA synthetase and tRNA(ser) in different organisms is reviewed. Secondly, using sequence alignments and phylogenetic trees, the early evolution of class 2 aminoacyl-tRNA synthetases is traced. Arguments are presented to suggest that synthetases are not the oldest of protein enzymes, but survived as RNA enzymes during the early period of the evolution of protein catalysts. In this view, the relatedness of the current synthetases, as evidenced by the division into two classes with their associated subclasses, reflects the replacement of RNA synthetases by protein synthetases. This process would have been triggered by the acquisition of tRNA 3' end charging activity by early proteins capable of activating small molecules (e.g., amino acids) with ATP. If these arguments are correct, the genetic code was essentially frozen before the protein synthetases that we know today came into existence.

Acylation↗
PubMed1998

Diagnosis of meningococcal meningitis by broad-range bacterial PCR with cerebrospinal fluid.

We used broad-range bacterial PCR combined with DNA sequencing to examine prospectively cerebrospinal fluid (CSF) samples from patients with suspected meningitis. Fifty-six CSF samples from 46 patients were studied during the year 1995. Genes coding for bacterial 16S and/or 23S rRNA genes could be amplified from the CSF samples from five patients with a clinical picture consistent with acute bacterial meningitis. For these patients, the sequenced PCR product shared 98.3 to 100% homology with the Neisseria meningitidis sequence. For one patient, the diagnosis was initially made by PCR alone. Of the remaining 51 CSF samples, for 50 (98.0%) samples the negative PCR findings were in accordance with the negative findings by bacterial culture and Gram staining, as well as with the eventual clinical diagnosis for the patient. However, the PCR test failed to detect the bacterial rRNA gene in one CSF sample, the culture of which yielded Listeria monocytogenes. These results invite new research efforts to be focused on the application of PCR with broad-range bacterial primers to improve the etiologic diagnosis of bacterial meningitis. In a clinical setting, Gram staining and bacterial culture still remain the cornerstones of diagnosis.

Cerebrospinal Fluid↗
PubMed1999

Expression of recombinant mouse sperm protein sp56 and assessment of its potential for use as an antigen in an immunocontraceptive vaccine.

Recombinant mouse sp56 protein was produced for testing as an antigen in an immunocontraceptive vaccine. The coding sequence for the mature sp56 protein was cloned into the bacterial expression system pFLAG using a PCR-based method on mouse testis cDNA. Polyclonal antisera were raised in mice against affinity purified recombinant sp56 fusion protein (sp56FLAG) or an artificial sp56 peptide fused to a carrier protein (KLH) and shown to cross-react to a protein band of 75 kD in detergent extracts of mouse sperm by Western immunoblot analysis under reducing conditions. The antisera to sp56FLAG also immunolocalized over the entire acrosome of mouse sperm. Female BALB/c mice were immunized intraperitoneally with sp56FLAG in a fertility trial with 20 microg sp56FLAG in Freund's Complete Adjuvant and boosted three to five times with 20 microg sp56FLAG in Freund's Incomplete Adjuvant. Litter sizes of sp56FLAG-treated mice were significantly smaller than control-treated animals after five boosts.

Amino Acid Sequence↗
PubMed1988

Structure and organization of Marchantia polymorpha chloroplast genome. IV. Inverted repeat and small single copy regions.

We characterized the genes in the regions of large inverted repeats (IRA and IRB, 10,058 base-pairs each) and a small single copy (SSC 19,813 bp) of chloroplast DNA from Marchantia polymorpha. The inverted repeat (IR) regions contain genes for four ribosomal RNAs (16 S, 23 S, 4.5 S and 5 S rRNAs) and five transfer RNAs (valine tRNA(GAC), isoleucine tRNA(GAU), alanine tRNA(UGC), arginine tRNA(ACG) and asparagine tRNA(GUU)). The gene organization of the IR regions in the liverwort chloroplast genome is conserved, although the IR regions are smaller (10,058 base-pairs) than any reported in higher plant chloroplasts. The small single-copy region (19,813 base-pairs) encoded genes for 17 open reading frames, a leucine tRNA(UAG) and a proline tRNA(GGG)-like sequence. We identified 12 open reading frames by homology of their coding sequences to a 4Fe-4S-type ferredoxin protein, a bacterial nitrogenase reductase component (Fe-protein), five human mitochondrial components of NADH dehydrogenase (ND1, ND4, ND4L, ND5 and ND6), two Escherichia coli ribosomal proteins (S15 and L21), two putative proteins encoded in the kinetoplast maxicircle DNA of Leishmania tarentolae (LtORF 3 and LtORF 4), and a bacterial permease inner membrane component (encoded by malF in E. coli or hisQ in Salmonella typhimurium).

Amino Acid Sequence↗
PubMed1975

Induction of base-pair substitution and frameshift mutations in wild-type and repair-deficient strains of Salmonella typhimurium by the photodynamic action of methylene blue.

Induction of back mutations to prototrophy by methylene blue (MB)-sensitized photodynamic (PD) treatment has been studied in wild-type and repair-deficient strains of Salmonella typhimurium carrying either the base-pair substitution mutation hisG46 or the frameshift mutation hisD30529 We found that reversion of the hisG46 mutation was increased in a strain carrying a uvrB deletion and decreased in a strain carrying a recA-type mutation. Reversion of the hisD3052 (frameshift) mutation, on the other hand, was decreased in both uvrB deletion and recA-type strains. The former results are consistent with the hypothesis that the majority of MB-sensitized PD-INDUCED BASE-PAIR SUBSTITUTION MUTATIONS ARIse by a mechanism similar to that currently believed to be involved in UV mutagenesis. The latter results suggest that PD-induced frameshift mutations may arise in some other way, and two possible mechanisms involving sequential action of the excision repair and recombinational repair pathways are considered.

Base Sequence↗
PubMed1992

Expression of the cDNA for NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase as a catalytically active enzyme in Escherichia coli.

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme involved in the catabolism of the prostaglandins. The cDNA for human placental 15-PGDH has been expressed in Escherichia coli as a catalytically active protein. The polymerase chain reaction was used to introduce restriction endonuclease sites at each end of the 15-PGDH coding sequence. The 15-PGDH DNA was then inserted into the bacterial expression plasmids pUC-18 and pUC-19 which contain the isopropyl-l-thio-beta-D-galactopyranoside (IPTG) inducible lacZ promoter. Extracts from E. coli containing these expression plasmids exhibited 15-PGDH activity which was inducible with (IPTG). Crude extracts from E. coli expressing 15-PGDH activity were found to contain proteins of the predicted sizes in stained SDS-polyacrylamide gels and in Western blots using human placental 15-PGDH antiserum. The specific activity in E. coli extracts was several hundred-fold higher than that seen in extracts from human placenta.

Amino Acid Sequence↗
PubMed2002

Importance of compartment formation for a self-encoding system.

A self-encoding system designed to have strict "compartition" of the molecules, i.e., to contain only a single molecule of DNA in each compartment, was established, and its evolutionary fate was analyzed. The system comprised the Thermus thermophilus DNA polymerase gene as the informational molecule and its protein product replicating the gene as the functional molecule. Imposing strict compartition allows the self-encoding system to last up to at least the tenth generation, whereas the system ceased to work after the third generation when loose compartition initiated with 100 molecules was imposed. These results provide experimental evidence on the importance of compartition for the maintenance of a self-encoding system. In addition, the extent of diversity in self-replication activity of the compartments was found to be another vital difference in the evolutionary dynamics between the strict and loose compartitions. Although the system with strict compartition provides widely diversified activity of the compartments at each generation, the values of the activity diverge only within a small range in the system with loose compartition. When the variety in the activity of a compartment is small, functional selection becomes weak, and to conform Darwinian evolution may become unfeasible. Therefore, strict compartition is essential for the evolvability of a self-encoding system.

Amino Acid Substitution↗
PubMed1974

Chemical carcinogens as frameshift mutagens: Salmonella DNA sequence sensitive to mutagenesis by polycyclic carcinogens.

Other investigators have shown that several polycyclic carcinogens are frameshift mutagens in Salmonella. Mutagenic potency of these compounds is assessed by ability to induce reversion of histidine-requiring frameshift mutants to prototrophy. One frameshift mutation in the histidinol dehydrogenase gene, hisD3052, is unusually sensitive to mutagenesis by certain polycyclic carcinogens. We find that the 3052 mutation is a - 1 deletion, probably loss of a G.C pair from a DNA repeat of [unk]. The polycyclic carcinogens tested (e.g., 2-nitroso-fluorene) revert 3052 by deleting a [unk] doublet from the DNA sequence [unk], which is close to the 3052 site. This rare mispairing-prone sequence represents the carcinogen-sensitive "hotspot" in 3052. The ICR compounds, noncarcinogenic intercalating agents, show a broader specificity of mutagenesis. Reversion with these mutagens occurs predominantly by two mechanisms: one identical to that of the polycyclic carcinogens, and the other by +1 additions in a third DNA tract narrowly separated from the 3052 site. The alkylating carcinogen N-methyl-N'-nitro-N-nitrosoguanidine appears to have a similar dual specificity. DNA base changes in 3052 revertants have been correlated with properties of histidinol dehydrogenase in crude extracts. This correlation of DNA base sequence with electrophoretic and other properties of the mutant proteins allows one to analyze easily the specificity of new mutagens that mutate this strain.

Acridines↗
PubMed2006

Cytosine usage modulates the correlation between CDS length and CG content in prokaryotic genomes.

Previous studies have argued that, given the AT-rich nature of stop codons, the length and CG% of coding sequences (CDSs) should be positively correlated. This prediction is generally supported empirically by prokaryotic genomes. However, the correlation is weak for a number of species, with 4 species showing a negative correlation. Here we formulate a more general hypothesis incorporating selection against cytosine (C) usage to explain the lack of strong positive correlation between the length and GC% of CDSs. Two factors contribute to the selection against C usage in long CDSs. First, C is the least abundant nucleotide in the cell, and a long CDS should have fewer Cs to increase transcription efficiency. Second, C is prone to mutation to U/T and selection for increased reliability should reduce C usage in long CDSs. Empirical data from prokaryotic genomes lend strong support for this new hypothesis.

Algorithms↗
PubMed2000

N-terminal mutations in the anti-estradiol Fab 57-2 modify its hapten binding properties.

Recombinant antibodies often contain N-terminal mutations arising from the use of degenerate cloning primer sets and/or the introduction of restriction sites in the framework 1 regions. We studied the effects of such mutations in a recombinant anti-estradiol Fab fragment derived from the hybridoma cell line 57-2. The 5' ends of the heavy and light chain genes were originally modified to introduce the restriction sites XhoI and SacI, respectively, for cloning purposes. However, the affinity and specificity of the recombinant Fab were lowered compared to the proteolytic Fab' fragment of the parental hybridoma IgG. Replacing the mutated sites with authentic amino acid coding sequences restored the binding properties as well as increased the bacterial production levels fivefold and 10-fold at 30 and 37 degrees C, respectively. Local changes in the antigen binding site were probed by determining the affinity constants (Kd) for estradiol and four related steroids. It was found that the mutated heavy chain amino terminus specifically increased the Kd for testosterone whereas the mutated light chain amino terminus decreased the Kd for all of the steroids to the same extent; the heavy and light chain effects were additive. Analysis of a newly determined crystal structure of the authentic Fab 57-2 in complex with estradiol suggests that mutations in the residue 2 in V(H), and 2 and 4 in the V(L) domain were those responsible for the observed effects. Their general roles as structure-determining residues for the CDR3 loops imply that similar effects can occur with other recombinant antibodies as well.

Amino Acid Substitution↗
PubMed1991

Arg encodes a widely expressed 145 kDa protein-tyrosine kinase.

Arg encodes a protein highly related to the c-abl gene product with regard to overall structural architecture as well as the amino acid sequences of their tyrosine kinase, and src-homologous 2 and 3 domains. The two genes form a distinct subfamily of non-receptor tyrosine kinases and share a common homolog in Drosophila. In this study we characterized the arg protein product by expression of its coding sequence in bacteria. The recombinant arg protein was detected in bacterial lysates by immunoblotting and exhibited a molecular mass of 145 kDa. Phosphoamino acid analysis of the arg gene product following an immune complex autokinase reaction revealed tyrosine phosphorylation and established that it possesses tyrosine kinase activity. High-titer antibody capable of detecting the cellular arg gene product was generated by expressing a carboxy-terminal segment of arg in bacteria and using the recombinant protein as an immunogen. The arg gene product was identified in cultured human cells as a 145 kDa protein that exhibited autokinase activity. Analysis of arg expression in murine tissues revealed that arg, like c-abl, is widely expressed, further extending the similarities between the two genes, and suggesting that arg probably functions in signaling pathways fundamental to many cell types.

DNA-Directed RNA Polymerases↗
PubMed1988

Operator constitutive mutation of 3-hydroxy-3-methylglutaryl coenzyme A reductase promoter abolishes protein binding to sterol regulatory element.

Through substitution mutagenesis we identified the promoter elements responsible for basal expression and sterol-mediated repression of transcription of the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-controlling enzyme of cholesterol biosynthesis. Mutant promoters containing 277 base pairs (bp) of reductase 5' flanking sequence were inserted into recombinant plasmids upstream of the coding region for bacterial chloramphenicol acetyltransferase. The plasmids were transfected into hamster fibroblasts, and transcription was measured in the presence and absence of sterols. Mutations in three regions that are known to bind nuclear proteins markedly reduced transcription. Mutation of another protein-binding region of 20 bp in length did not reduce transcription, but it did abolish sterol-mediated repression, producing an operator constitutive phenotype. This mutation also abolished protein binding to the corresponding 20-bp region of DNA as determined by footprinting assays. When a DNA fragment containing these 20 bp was inserted into the herpes simplex virus thymidine kinase promoter, sterol-mediated repression was observed. This sequence contains an octanucleotide that shows a 7/8-bp match with a previously identified regulatory sequence in repeat 2 of the low density lipoprotein receptor promoter, another sterol-repressible gene. We hypothesize that this octanucleotide, GTGGCGGTG, is the core binding site for a sterol-dependent protein that represses transcription.

Animals↗
PubMed2000

A recombinant rat regenerating protein is mitogenic to pancreatic derived cells.

Pancreatic regenerating protein (reg I) is expressed in acinar cells and is mitogenic to beta- and ductal cells. Isolation of large amounts of endogenous reg I for in vivo and in vitro experiments has been difficult. The aim of this study was to develop a recombinant protein and determine its bioactivity on rat pancreatic derived cells. cDNA of the rat reg I coding region was created with unique BamHI flanking sequences using reverse transcriptase PCR. The coding region was then cloned into a bacterial expression vector in which expression is controlled by a T7 promoter. After transformation into the Escherichia coli strain B21(DE3) and induction by isopropyl-beta-d-thiogalactopyranoside, a fusion protein of 24 kDa in size, named reg-PET, was noted in the bacterial lysate. This protein contained a polyhistidine and S-peptide sequence to facilitate isolation and identification, respectively. This protein was purified using affinity chromatography, and identity was confirmed with gel electrophoresis and Western analysis. The reg-PET protein was mitogenic to both ARIP and RIN cells, rat pancreatic ductal and beta-cell lines, respectively. Antibodies raised to the protein reacted against rat reg I in pancreas. The purified recombinant reg I fusion protein, like endogenous reg I, is mitogenic to pancreatic derived cells. It is more potent than reg I isolated from pancreatic tissue. This protein can be isolated rapidly, easily, and with a high amount of purity.

Amino Acid Sequence↗
PubMed2006

Identification of circular codes in bacterial genomes and their use in a factorization method for retrieving the reading frames of genes.

We developed a statistical method that allows each trinucleotide to be associated with a unique frame among the three possible ones in a (protein coding) gene. An extensive gene study in 175 complete bacterial genomes based on this statistical approach resulted in identification of 72 new circular codes. Finding a circular code enables an immediate retrieval of the reading frame locally anywhere in a gene. No knowledge of location of the start codon is required and a short window of only a few nucleotides is sufficient for automatic retrieval. We have therefore developed a factorization method (that explores previously found circular codes) for retrieving the reading frames of bacterial genes. Its principle is new and easy to understand. Neither complex treatment nor specific information on the nucleotide sequences is necessary. Moreover, the method can be used for short regions in nucleotide sequences (less than 25 nucleotides in protein coding genes). Selected additional properties of circular codes and their possible biological consequences are also discussed.

Base Sequence↗
PubMed1997

Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.

The nucleotide sequence encoding the N-terminal domain (residues 1-183) of human apolipoprotein E3 (apoE3) was cloned into the pET expression vector and introduced into Escherichia coli. Induction of protein expression with isopropyl beta-D-thiogalactopyranoside resulted in production of recombinant apoE3(1-183). Immunoblot analysis revealed that recombinant protein was present in both the cell pellet and cell culture supernatant. Analysis revealed that a significant portion of the rApoE3(1-183) in the cell pellet still possessed the bacterial N-terminal pel B leader sequence, encoded by plasmid DNA directly upstream of the apoE3(1-183) coding sequence. By contrast, this hydrophobic leader sequence had been removed from recombinant protein specifically accumulating in the culture medium. This behavior is novel for bacterial expression of apolipoprotein E and its truncated variants and permits efficient overexpression of the recombinant protein (> 100 mg/L cell culture). Recombinant apoE3(1-183) was isolated by a combination of heparin-Sepharose chromatography and reverse-phase HPLC. Electrospray mass spectrometry provided a mass of 21 191 daltons, corresponding directly to that expected from the known sequence. Circular dichroism spectroscopy revealed that the recombinant protein possesses significant amounts of alpha-helical secondary structure. The lipid binding ability of rApoE3(1-183) was evaluated using an in vitro lipoprotein binding assay. It was observed that recombinant apoE3(1-183) protected human low density lipoprotein (LDL) from lipid accumulation induced particle aggregation, indicating that it is capable of associating with lipoprotein surfaces. In addition, rApoE3(1-183) forms disk complexes with the model phospholipid dimyristoylphosphatidylcholine. In competition experiments, it was observed that rApoE3(1-183) phospholipid disks compete with 125I-LDL for binding to the apoB/E receptor on human skin fibroblasts to an extent similar to that observed for intact rApoE3. Taken together, these data show that recombinant apoE3(1-183) is fully functional as an apolipoprotein and receptor ligand. Given the high expression level and its known existence as a monomer in solution, we evaluated the potential for application of NMR spectroscopy to study the structure-function relationship of rApoE3(1-183). Bacteria were cultured in media supplemented with 15NH4Cl or [15N]glycine and the isotopically labeled recombinant apoE3(1-183) was analyzed by heteronuclear single quantum correlation NMR spectroscopy. The data revealed that rApoE3(1-183) is an excellent candidate for solution structure studies by NMR, including conformational adaptations associated with lipid association.

Apolipoprotein E3↗
PubMed2003

Cloning of modular type I polyketide synthase genes from salinomycin producing strain of Streptomyces albus.

Cloning of polyether polyketide synthase (PKS) genes for salinomycin biosynthesis was attempted from Streptomyces albus. Seven beta-ketoacyl synthase (KS) core regions were obtained by PCR amplification using primers designed based on the conserved KS domains of type I PKSs. Using the KS fragment as a probe, screening of an S. albus genomic DNA library was carried out by colony hybridization. From the positive cosmid clone isolated, a 4.5-kbBamHI fragment was subcloned and sequenced. It showed high homology with bacterial type I PKSs and was deduced to code for KS, malonyl transferase, and ketoreductase motifs. By gene disruption with this 4.5-kb BamHI fragment, the cloned gene was shown to be a part of the salinomycin biosynthetic gene cluster of S. albus.

Amino Acid Sequence↗
PubMed1987

Efficient integrative transformation of Cephalosporium acremonium.

A hybrid gene, IPNSp/HPTorf, was constructed by placing an 850 bp sequence of Cephalosporium acremonium DNA next to the 5' end of a bacterial open reading frame, HPTorf. The sequence was obtained as an 850 bp NcoI restriction fragment from the 5' non-coding region of the C. acremonium isopenicillin N synthetase (IPNS) gene. The HPTorf was obtained from a bacterial gene that coded for a hygromycin B phosphotransferase (HPT). Plasmids that contained IPNSp/HPTorf transformed C. acremonium to a stably maintained hygromycin B resistant phenotype. Southern analysis of total DNA from transformants demonstrated multiple integrations of the transforming DNA in the high molecular weight DNA of most transformants, but single integrations were observed in a few transformants. The number of transformants per microgram of DNA was about 100 times greater than for plasmids that contained the HPTorf without any juxtaposed eucaryotic promoter sequence. Plasmids with the promoterless HPTorf and plasmids with a truncated S. cerevisiae phosphoglycerate kinase promoter juxtaposed to the HPTorf transformed C. acremonium at equivalent low frequencies. Transformation of C. acremonium with linearized plasmid DNA produced at least 2-3 fold more transformants than the corresponding circular molecule. Several observations were made concerning protoplast formation and handling which made the transformation procedure more efficient and allowed a greater proportion of protoplasts to regenerate to viable walled cells. Plasmids were constructed that contained both the IPNSp/HPTorf and additional elements: fragments of C. acremonium ribosomal DNA (rDNA), or a fragment of C. acremonium mitochondrial DNA possessing activity as an autonomous replication sequence (ARS) in S. cerevisiae, or putative transcriptional termination/polyadenylation signals from the IPNS gene. These plasmids transformed C. acremonium at frequencies experimentally equivalent to those containing IPNSp/HPTorf without any of these additional elements.

Acremonium↗