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PubMed1988

Transcription in the region of the replication origin, oriC, of Escherichia coli: termination of asnC transcripts.

Transcription from the asnC promoter was found to proceed through the replication origin, oriC, into the gidA gene of Escherichia coli. Between 10% and 20% of asnC transcripts reached oriC in vivo. Termination sites in the intergenic region between asnC and mioC and within mioC were determined in vivo and in vitro using S1 mapping. Only about 10% of the transcripts terminated at the asnC terminator in vivo. DnaA protein dependent termination was observed close to the binding site, dnaA box, for DnaA protein. In the in vitro replication system asnC transcripts did not reach oriC, suggesting that asnC transcripts are not involved in the initiation of replication, contrary to mioC transcripts. We suggest that oriC and mioC might have been transposed during evolution into an asnC regulation.

Bacterial Proteins↗
PubMed1985

Gene amplification: an example of accelerated evolution in tumorigenic cells.

During selection for methotrexate resistance, tumorigenic CHEF/16 cells and derivatives from CHEF/16 tumors underwent amplification of the dihydrofolate reductase gene (DHFR) at accelerated rates compared with closely related nontumorigenic CHEF/18 cells. "Dot blot" analysis showed that the CHEF/16 cells contained many more copies of the DHFR gene than did the CHEF/18 cells, when assayed at similar elevated levels of methotrexate resistance. Chromosome analysis of cell samples taken at several time points during amplification revealed large differences between the nontumorigenic CHEF/18 cells and the two tumorigenic cell lines. The tumorigenic cells developed few chromosome rearrangements over a 4-log increase in methotrexate resistance, other than increased length of a single chromosome, which was shown by in situ hybridization to contain most or all of the amplified DHFR gene copies. In contrast, the CHEF/18 cells underwent complex, progressive changes in almost every chromosome, and in situ hybridization suggested a dispersed pattern of gene amplification. The data support the hypothesis that unregulated amplification is a pathological process, occurring readily in neoplastic but rarely in normal cells, that together with other chromosomal disturbances contributes to the rapid evolution and progression of cancer.

Animals↗
PubMed2005

Loss of editing activity during the evolution of mitochondrial phenylalanyl-tRNA synthetase.

Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNA(Phe). Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNA(Phe), an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol.

Adenosine Triphosphate↗
PubMed1995

CCAAT-box binding protein NF-Y (CBF, CP1) recognizes the minor groove and distorts DNA.

The CCAAT box is one of the most common promoter elements. The evolutionarily conserved heteromeric factor NF-Y binds this sequence with high affinity and specificity. By comparing the methylation interference patterns of different sites, performing electrophoretic mobility shift assays (EMSA) with IC-substituted oligonucleotides and competition experiments with the minor groove binding (MGB) drugs distamicin A, tallimustine and Hoechst 33258 we show that NF-Y makes key minor groove interactions. Circular permutation assays on four CCAAT boxes, MHC Class II Ea, HSP70, epsilon-globin and MSV, indicate that NF-Y is able to distort the double helix by angles of 62-82 degrees, depending on the site used, and suggest that nucleotides flanking the CCAAT pentanucleotide influence the degree of bending.

Base Sequence↗
PubMed2001

Expression and stabilization of galactose oxidase in Escherichia coli by directed evolution.

We have used directed evolution methods to express a fungal enzyme, galactose oxidase (GOase), in functional form in Escherichia coli. The evolved enzymes retain the activity and substrate specificity of the native fungal oxidase, but are more thermostable, are expressed at a much higher level (up to 10.8 mg/l of purified GOase), and have reduced negative charge compared to wild type, all properties which are expected to facilitate applications and further evolution of the enzyme. Spectroscopic characterization of the recombinant enzymes reveals a tyrosyl radical of comparable stability to the native GOase from Fusarium.

Amino Acid Motifs↗
PubMed1994

Influence of a steroid receptor DNA-binding domain on transcriptional regulatory functions.

We have isolated two independent mutations in the DNA-binding domain of the rat glucocorticoid receptor, P493R and S459A, that implicate DNA binding in the control of attached transcriptional activation domains, either that of the receptor itself or of VP16. The mutants are capable of activating transcription normally, but unlike wild-type receptors, they interfere with particular transcriptional activators in yeast and mammalian cells, and inhibit growth when overexpressed in yeast. The mutant residues reside at positions within the three-dimensional structure of the receptor that could, in principle, transduce structural changes from the DNA-binding surface of the receptor to other functional domains. These findings, together with the salt dependence of specific and nonspecific DNA binding by these receptors, suggest that specific DNA acts as an allosteric effector that directs the functional interaction of the receptor with targets of transcriptional activation and that the P493R and S459A mutants mimic the allosteric effect of specific DNA, allowing the receptor to interact with regulatory targets even in the absence of specific DNA binding.

Allosteric Regulation↗
PubMed2004

Intraspecies sequence comparisons for annotating genomes.

Analysis of sequence variation among members of a single species offers a potential approach to identify functional DNA elements responsible for biological features unique to that species. Due to its high rate of allelic polymorphism and ease of genetic manipulability, we chose the sea squirt, Ciona intestinalis, to explore intraspecies sequence comparisons for genome annotation. A large number of C. intestinalis specimens were collected from four continents, and a set of genomic intervals were amplified, resequenced, and analyzed to determine the mutation rates at each nucleotide in the sequence. We found that regions with low mutation rates efficiently demarcated functionally constrained sequences: these include a set of noncoding elements, which we showed in C. intestinalis transgenic assays to act as tissue-specific enhancers, as well as the location of coding sequences. This illustrates that comparisons of multiple members of a species can be used for genome annotation, suggesting a path for the annotation of the sequenced genomes of organisms occupying uncharacterized phylogenetic branches of the animal kingdom. It also raises the possibility that the resequencing of a large number of Homo sapiens individuals might be used to annotate the human genome and identify sequences defining traits unique to our species.

Animals↗
PubMed1989

The design and catalytic properties of a simplified ribonuclease P RNA.

Ribonuclease P (RNase P) RNA is the catalytic moiety of the ribonucleoprotein enzyme that removes precursor sequences from the 5' ends of pre-transfer RNAs in eubacteria. Phylogenetic variation according to recently proposed secondary structure models was used to identify structural elements of the RNase P RNA that are dispensable for catalysis. A simplified RNase P RNA that consists only of evolutionarily conserved features was designed, synthesized, and characterized. Although the simplified RNA (Min 1 RNA) is only 263 nucleotides in length, in contrast to the 354 to 417 nucleotides of naturally occurring RNase P RNAs, its specificity of pre-tRNA cleavage is identical to that of the native enzymes. Moreover, the catalytic efficiencies of the Min 1 RNA and the native RNA enzymes are similar. These results focus the search for the catalytic elements of RNase P RNAs to their conserved structure.

Bacillus megaterium↗
PubMed2000

Genetic organization of the downstream region of the mecA element in methicillin-resistant Staphylococcus aureus isolates carrying different polymorphisms of this region.

We describe here the genetic organization of the mec element downstream of the mecA gene in 34 different methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates carrying 13 of the most frequent polymorphisms of mecA and representing the major epidemic clones of MRSA. All polymorphisms carried three common genetic elements: the hypervariable region, a copy of IS431, and a unique 2-kb sequence (downstream constant segment, or dcs) for which no homologous sequences are found in data banks. Polymorphisms of the downstream region were shown to be caused by the presence of linearized plasmids flanked by insertion sequences (pUB110, pT181, and pI258) and the autonomous insertion sequence IS256.

Bacterial Proteins↗
PubMed1993

Use of an Exotic Carbon Source To Selectively Increase Metabolic Activity and Growth of Pseudomonas putida in Soil.

Respiration and growth of Pseudomonas putida PpG7, containing catabolic plasmid NAH7, was determined in three agricultural field soils amended with the carbon source salicylate. The addition of salicylate to soil significantly increased the population of PpG7. However, there was a lack of relationship between microbial numbers and activity as determined by evolution of CO(2). In soils containing 30 to 1,500 mug of salicylate per g, metabolic activities of PpG7 peaked between 18 and 42 h and population densities increased approximately 10-to 10-fold. However, the metabolic activity of PpG7 rapidly declined after salicylate was utilized, whereas peak population densities were maintained for the duration of the experiments (5 to 7 days). Thus, elevated population densities of PpG7 were represented by inactive cells. Soil type had only minor effects on respiration rates or growth curves of PpG7 when amended with comparable concentrations of salicylate. Respiration and growth rates were optimal at concentrations between 300 and 1,000 mug of salicylate per g in the test soils. At 1,500 to 2,500 mug/g, respiration and growth of PpG7 were initially suppressed, but after a short lag time both attained levels similar to or greater than those resulting from the use of lower concentrations of salicylate. The culturing of PpG7 on a salicylate-amended medium to induce salicylate-degradative enzymes did not affect the lag time before utilization of salicylate in soil. Although PpG7 competed well with fungi for the substrate, suppression of fungal populations with cycloheximide resulted in significantly increased population densities of PpG7 in two of three soils amended with salicylate. The beneficial activities of bacteria in soil are discussed in relation to population density, population metabolic activity, and selective carbon source utilization.

Journal Article↗
PubMed1994

In vivo evolution of c-rel oncogenic potential.

The c-rel proto-oncogene belongs to the NF-kappa B/rel and I kappa B gene families, which regulate several inducible processes, including self-defense/repair and embryogenesis. Transduction of the c-rel transcription factor by the avian retrovirus resulted in the formation of a highly oncogenic virus, reticuloendotheliosis virus strain T (REV-T), that encodes the oncogene v-rel. To examine the oncogenic potential of c-rel, we inserted it into a REV-T-based retroviral vector, rescued virus [REV-C(CSV)], and infected 1-day-old chicks. All birds developed tumors, and all cell lines established from REV-C-induced tumors expressed c-rel proteins that lacked C-terminal sequences. These proteins, responsible for both in vivo and in vitro cell proliferation, were apparently selected for their oncogenic potential. In order to examine the cooperation of C-terminal deletions with other oncogenic alterations in vivo, point mutations present in the N-terminal and middle regions of v-rel were analyzed by a similar protocol. The data obtained support four conclusions. (i) c-rel proteins bearing any of three single-amino-acid mutations present in the N-terminal portion of v-rel were sufficiently oncogenic to induce tumor development in the absence of additional mutations. (ii) Combining a mutation from the N-terminal region of v-rel with a deletion of the C-terminal sequences of c-rel increases the oncogenicity of the protein in an additive manner. (iii) Mutations present in the middle of v-rel cooperated synergistically with C-terminal deletions to produce highly transforming viruses. (iv) Deletion of c-rel produced a variety of transforming rel proteins with sizes that extended from 42 to 65 kDa. The most frequently isolated rel deletion was 62 kDa in size. To examine the basis for the selection of different rel mutants, their ability to induce immunoregulatory surface receptors was analyzed. The data revealed a correlation between the induction capacity of these mutants and their corresponding contribution to in vivo tumorigenic potential. Moreover, an analysis of the subcellular localization of different rel proteins revealed an inverse correlation between the size of the protein and the proportion in the nucleus of lymphoid cells.

Alpharetrovirus↗
PubMed2005

An acquisition account of genomic islands based on genome signature comparisons.

BACKGROUND: Recent analyses of prokaryotic genome sequences have demonstrated the important force horizontal gene transfer constitutes in genome evolution. Horizontally acquired sequences are detectable by, among others, their dinucleotide composition (genome signature) dissimilarity with the host genome. Genomic islands (GIs) comprise important and interesting horizontally transferred sequences, but information about acquisition events or relatedness between GIs is scarce. In Vibrio vulnificus CMCP6, 10 and 11 GIs have previously been identified in the sequenced chromosomes I and II, respectively. We assessed the compositional similarity and putative acquisition account of these GIs using the genome signature. For this analysis we developed a new algorithm, available as a web application. RESULTS: Of 21 GIs, VvI-1 and VvI-10 of chromosome I have similar genome signatures, and while artificially divided due to a linear annotation, they are adjacent on the circular chromosome and therefore comprise one GI. Similarly, GIs VvI-3 and VvI-4 of chromosome I together with the region between these two islands are compositionally similar, suggesting that they form one GI (making a total of 19 GIs in chromosome I + chromosome II). Cluster analysis assigned the 19 GIs to 11 different branches above our conservative threshold. This suggests a limited number of compositionally similar donors or intragenomic dispersion of ancestral acquisitions. Furthermore, 2 GIs of chromosome II cluster with chromosome I, while none of the 19 GIs group with chromosome II, suggesting an unidirectional dispersal of large anomalous gene clusters from chromosome I to chromosome II. CONCLUSION: From the results, we infer 10 compositionally dissimilar donors for 19 GIs in the V. vulnificus CMCP6 genome, including chromosome I donating to chromosome II. This suggests multiple transfer events from individual donor types or from donors with similar genome signatures. Applied to other prokaryotes, this approach may elucidate the acquisition account in their genome sequences, and facilitate donor identification of GIs.

Algorithms↗
PubMed1989

Genomic organization and polymorphisms of the human C3d/Epstein-Barr virus receptor.

The human C3d/Epstein-Barr virus receptor (CR2/CD21) is a 145-kDa protein primarily expressed on mature B lymphocytes. CR2 is a member of the regulators of complement activation (RCA) gene family found on band q32 of chromosome 1. The RCA proteins are characterized by the presence of 60-70 amino acid short consensus repeats (SCR). A full length CR2 cDNA was cloned and used to identify overlapping cosmid genomic clones. Analysis of CR2 exon-intron junctions revealed the presence of three types of exons in the short consensus repeat region of CR2. First, four exons each of which encodes two SCR are present. Five exons encode a single SCR. Six exons encode SCRs which are split in identical positions. The order of these types of exons is in a repeated array of four SCRs, indicating that the contemporary CR2 gene likely evolved from a more primitive gene containing four SCRs. The CR2 full length cDNA clone was used to find restriction fragment length polymorphisms (RFLPs). Restriction enzyme TaqI generated 2.55- and 2.10-kilobase (kb) polymorphic bands. This RFLP was mapped near the exon containing the first two SCRs. HaeIII digestion generated polymorphic bands of 1.45, 1.55, and 1.75 kb. The HaeIII 1.45-kb RFLP band maps near the exon containing the 15th SCR. The TaqI and HaeIII RFLPs will provide tools for the genetic analysis of CR2. The organization of the CR2 gene provides insights into the evolution of human CR2 and the RCA gene family.

Amino Acid Sequence↗
PubMed1988

Nucleotide sequence and expression of rabbit globin genes zeta 1, zeta 2, and zeta 3. Pseudogenes generated by block duplications are transcriptionally competent.

The addition of two embryonic globin genes, zeta 0 and zeta 4, to the rabbit alpha-like globin gene cluster expands it to include eight genes arranged 5'-zeta 0-zeta 1-alpha 1-theta 1-zeta 2-zeta 3-theta 2-zeta 4-3'. The identification of these new genes supports the model that this gene cluster evolved by a series of block duplications of gene sets. The nucleotide sequence of three embryonic zeta-globin genes, zeta 1, zeta 2, and zeta 3, shows that all three are pseudogenes. Gene zeta 1 contains two frame-shift deletions, gene zeta 2 has lost exon 1 as well as the 5' promoter sequences, and gene zeta 3 has lost and replaced codons for amino acids that are critical for the function of alpha- and zeta-globin polypeptides. However, genes zeta 1 and zeta 3 are still transcriptionally competent, as shown by the accurate initiation and processing of transcripts from cloned genes introduced into HeLa cells. A quantitative comparison of the zeta-globin gene sequences indicates that the ancestor to the rabbit zeta 1 and zeta 3 genes was inactivated about 44 million years ago, and the block duplication that formed the two genes occurred about 28 million years ago. About 600 base pairs of the 5'-flanking sequence of the rabbit zeta 3-globin gene is very similar to the 5' flanks of three other mammalian zeta-globin genes.

Animals↗
PubMed1988

Evolutionary conservation of the product of human c-myc exon 1 and its inducible expression in a murine cell line.

The human c-myc proto-oncogene contains an open reading frame within its first exon which is translated into protein (MYCHEX1). While the murine c-myc exon 1 is obviously non coding, we show that in mouse cells there are polypeptides closely related to human MYCHEX1. These polypeptides share the same immunological reactivities with the human polypeptides. Furthermore, the 32 kDa polypeptide of murine cells has, like its human counterpart, the ability to dimerise in a 58 kDa form in denaturing and reducing SDS-PAGE. The human gene was introduced into a murine cell line by transfection. A cell line was studied, in which the inducible expression of the gene allows a substantial increase in the concentration of the corresponding protein. This inducible protein behaves in any respect like the murine one, either in SDS-PAGE or in a specific immunoassay. These shared properties constitute a further proof that the human and mouse MYCHEX1 proteins are encoded by the sequence overlapping the human myc exon 1 and a related murine sequence. The gene contained in the human c-myc exon 1 is not, therefore, a specific feature of human cells.

Animals↗
PubMed1994

[Antibiotic resistance of Campylobacter strains and its role in evolutionary processes in bacteria of the genus Campylobacter].

The influence of multiple antibiotic resistance of Campylobacter strains determined by R plasmids on their virulence was studied. It was shown that the strains with multiple resistance were mostly isolated from children with campylobacteriosis (26.5 per cent). The number of such strains isolated from the infected adults, hens and environmental objects amounted to 25.8, 23.3 and 21.4 per cent respectively. The difference of the resistance determinants in the tested strains was statistically insignificant. It was suggested that the R plasmids of the human strains could be as well detected in the strains from the infected hens and environmental objects. A chromosome-plasmid pattern of the Campylobacter resistance to kanamycin, tetracycline and erythromycin was determined. The analysis of the cytopathogenic activity of the plasmid-containing strains and their aplasmid clones revealed that this criterion of the virulence statistically significantly increased after the plasmid loss by the strains. It was concluded that the antibiotic resistant strains had a selective superiority while circulating in various ecological niches.

Adult↗
PubMed1996

[Analysis of expression of foreign genetic material under the control of heterogeneous promoters in the process of early development of the loach (Misgurnus fossilis L.)].

Promoters normally active in the nuclei of evolutionarily distant from fishes taxa-bacteria, insects, and Protozoa-are shown to be able to regulate heterologous genes during early development of the loach. It is shown that particular DNA sequences can drastically influence the function of eukaryotic promoters, enhancing the expression level.

Animals↗