Screening and selection strategies for disulfide isomerase activity.
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Cloning, Molecular↗
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Results for “plasmid evolution”
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[Towards a new bacteriology].
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Bacteria↗
[Nucleotide sequences of 5S rRNA genes of polyploid species of wheat and Aegilops species].
Primary structures of 5S rRNA genes and of non-transcribed spacers between them were determined in families of 5S DNA repeats 420 and 500 b.p. long in 8 wheat and Aegilops species. The high conservatism of sequences coding for 5S rRNA, 3'- and 5'-ends of non-transcribed spacers was shown not to depend on the evolutional position, ploidy level and genomic composition of species. The activity of transcription of 5S rRNA cloned genes was determined in vitro. The functional heterogeneity was revealed in each family of repeats due to the existence of exchanges of separate nucleotides within the internal transcription control region. A greater deficiency of CpG dinucleotide was revealed in 5S rRNA genes than in non-transcribed spacers.
Base Sequence↗
The DNA sequence and minimal replicon of the Corynebacterium glutamicum plasmid pSR1: evidence of a common ancestry with plasmids from C. diphtheriae.
The complete nucleotide sequence of pSR1, a 3 kb multicopy cryptic plasmid from Corynebacterium glutamicum ATCC 19223 has been determined. pSR1 is unrelated to the 4.4 kb Brevibacterium lactofermentum plasmid pBL1 and shows no DNA sequence conservation with plasmids from Staphylococcus. Transposon insertion and deletion mutants located the minimal replicon to within a 2.1 kb NcoI-BclI restriction fragment. This region contains a single large open reading frame, ORF2, flanked at the 5' end by a series of inverted repeat sequences which may modulate its expression, and at the 3' end by a region which may contain a replication origin. ORF2 (position 1633-2636) with a maximum coding potential of 36 kDa is essential for pSR1 replication and was designated the rep gene. The predicted ORF2 protein product exhibits 47% identity over a length of 343 amino acids with a replication-associated ORF in the C. diphtheriae plasmid pNG2, many of the changes being in the third base position. This observation suggests that pSR1 and pNG2, which are two plasmids from environmentally separated Corynebacterium species, may share a common ancestral rep gene.
Amino Acid Sequence↗
DNA repair and the evolution of transformation in the bacterium Bacillus subtilis.
The purpose of the work reported here is to test the hypothesis that natural genetic transformation in the bacterium Bacillus subtilis has evolved as a DNA repair system. Specifically, tests were made to determine whether transformation functions to provide DNA template for the bacteria] cell to use in recombinational repair. The survivorship and the homologous transformation rate as a function of dose of ultraviolet irradiation (UV) was studied in two experimental treatments, in which cells were either transformed before (DNA-UV), or after (UV-DNA), treatment with UV. The results show that there is a qualitative difference in the relationship between the survival of transformed cells (sexual cells) and total cells (primarily asexual cells) in the two treatments. As predicted by the repair hypothesis, in the UV-DNA treatment, transformed cells had greater average survivorship than total cells, while in the DNA-UV treatment this relationship was reversed. There was also a consistent and qualitative difference between the UV-DNA and DNA-UV treatments in the relationship between the homologous transformation rate (transformed cells/total cells) and UV dosage. As predicted by the repair hypothesis, the homologous transformation rate increases with UV dose in the UV-DNA experiments but decreases with UV dose in the DNA-UV treatments. However, the transformation rate for plasmid DNA does not increase in a UV-DNA treatment. These results support the DNA repair hypothesis for the evolution of transformation in particular, and sex generally.
Bacillus subtilis↗
Rapid evolution of novel traits in microorganisms.
The use of natural microorganisms in biotransformations is frequently constrained by their limited tolerance to the high concentrations of metabolites and solvents required for effective industrial production. In many cases, more robust strains have to be generated by random mutagenesis and selection. This process of directed evolution can be accelerated in mutator strains, which carry defects in one or more of their DNA repair genes. However, in order to use mutator strains, it is essential to restore the normal low mutation rate of the selected organisms immediately after selection to prevent the accumulation of undesirable spontaneous mutations. To enable this process, we constructed temperature-sensitive plasmids that temporarily increase the mutation frequency of their hosts by 20- to 4,000-fold. Under appropriate selection pressure, microorganisms transformed with mutator plasmids can be quickly evolved to exhibit new, complex traits. By using this approach, we were able to increase the tolerance of three bacterial strains to dimethylformamide by 10 to 20 g/liter during only two subsequent transfers. Subsequently, the evolved strains were returned to their normal low mutation rate by curing the cells of the mutator plasmids. Our results demonstrate a new and efficient method for rapid strain improvement based on in vivo mutagenesis.
Dimethylformamide↗
Karyotype evolution in a simian virus 40-transformed tumorigenic human cell line.
Normal human foreskin fibroblasts (HSF4) were transfected using the pSV3-neo plasmid. A pool of 10 G418-resistant colonies, HSF4-T12, showed a progressive increase in the expression of a number of in vitro transformation markers with passage in culture and became immortalized. Although no tumors were formed when cells were injected subcutaneously into nude mice, this cell line produced progressive tumors when cells were injected into preimplanted Gelfoam sponges in the mice. When these tumors were cultured in vitro and subsequently injected subcutaneously, progressive tumors were produced with median latency periods as short as 4 weeks. Three phases of cytogenetic change could be distinguished. At early passages after transfection. HSF4-T12 exhibited many random chromosomal changes. At a time just after immortalization, both flow karyotype and G-banded analyses showed the appearance of balanced clonal rearrangements. These included t(2;4), t(2;14), t(3;?), 6p-, i(6p), 8p-, t(14;15), i(15), and t(18;?). These clonal rearrangements were stable with passage in culture, and less variability from cell to cell was noted. The only consistent chromosomal loss observed was -Y. Analysis of three independent tumors showed characteristic loss of chromosomal material rather than balanced chromosomal rearrangements. Frequent loss of 6q and chromosomes #13, 15, 20, and Y was noted.
Animals↗
Genetic content and evolution of adenoviruses.
This review provides an update of the genetic content, phylogeny and evolution of the family Adenoviridae. An appraisal of the condition of adenovirus genomics highlights the need to ensure that public sequence information is interpreted accurately. To this end, all complete genome sequences available have been reannotated. Adenoviruses fall into four recognized genera, plus possibly a fifth, which have apparently evolved with their vertebrate hosts, but have also engaged in a number of interspecies transmission events. Genes inherited by all modern adenoviruses from their common ancestor are located centrally in the genome and are involved in replication and packaging of viral DNA and formation and structure of the virion. Additional niche-specific genes have accumulated in each lineage, mostly near the genome termini. Capture and duplication of genes in the setting of a 'leader-exon structure', which results from widespread use of splicing, appear to have been central to adenovirus evolution. The antiquity of the pre-vertebrate lineages that ultimately gave rise to the Adenoviridae is illustrated by morphological similarities between adenoviruses and bacteriophages, and by use of a protein-primed DNA replication strategy by adenoviruses, certain bacteria and bacteriophages, and linear plasmids of fungi and plants.
Adenoviridae↗
Kinetics of growth and metabolism of Clostridium thermosaccharolyticum culture. Isolation and characteristics of its plasmids.
The kinetics of growth and metabolism of Clostridium thermosaccharolyticum DSM 571 has been studied at varying initial pH and glucose concentration. A weak inhibitory effect of excess glucose on the specific growth rate has been shown. The effect of antibiotics of various classes on culture growth and hydrogen evolution has been studied. Streptomycin and kanamycin resistance of this culture has been revealed as well as the phenomenon of increased hydrogen production in the presence of the above antibiotics. New plasmids, pNB1 (4.9 kb) and pNB2 (2.0 kb), were isolated from C. thermosaccharolyticum DSM 571. The restriction analysis of pNB1 and pNB2 has been performed.
Anti-Bacterial Agents↗
Nucleotide sequence of cloned rat serum albumin messenger RNA.
The nucleotide sequences of the recombinant DNA inserts of three bacterial plasmid clones containing nearly all of the rat serum albumin mRNA have been determined. A statistical analysis of the nucleotide sequence reveals a pattern of repeated internal homology that confirms the "intragenic triplication" model of albumin evolution.
Amino Acid Sequence↗
Carotenoid desaturases from Rhodobacter capsulatus and Neurospora crassa are structurally and functionally conserved and contain domains homologous to flavoprotein disulfide oxidoreductases.
The characteristic red color of some photosynthetic bacteria and the orange color of Neurospora conidia is due to the presence of carotenoids, photoprotective pigments synthesized by plants, algae, bacteria, and fungi. Generally, carotenoids are tetraterpenes in which absorption of visible light and photoprotection are mediated by a chain of conjugated double bonds, the chromophore, which is formed by successive desaturations of phytoene, a colorless precursor. The genes al-1 and crtI mediate the desaturation of phytoene in Neurospora crassa and Rhodobacter capsulatus, respectively. Here, we report that alignment of the primary sequence of Al-1, CrtI, and CrtD, another carotenoid desaturase, reveals conservation with amino acid residues that mediate FAD-binding and dimerization functions in Azotobacter vinelandii dihydrolipoamide dehydrogenase and human glutathione reductase, two disulfide oxidoreductases. Plasmids containing the coding region of an al-1 cDNA fused to appropriate bacterial transcriptional and translational signals complement crtI mutants. Our results indicate that both structure and function of carotenoid desaturases have been conserved during evolution and suggest that these enzymes are evolutionarily related to disulfide oxidoreductases.
Amino Acid Sequence↗
Plasmid fingerprinting. A tool for bacterial strain identification and surveillance of nosocomial and community-acquired infections.
Plasmid fingerprinting provides a rapid and dependable means of identifying bacterial isolates of the same strain. The stability, wide distribution, and diverse nature and size of extrachromosomal elements make it suitable for virtually all bacterial genera. There are many different procedures available for plasmid screening, and the one chosen depends primarily on the types of organisms to be analyzed. Some procedures are better suited to gram-positive organisms; others are better for visualizing the very large plasmids often seen in Pseudomonas and Rhizobium species. The key to the plasmid fingerprinting technique is agarose gel electrophoresis. In this step of the technique, it is important to differentiate open circular from closed circular forms of plasmids and to recognize the "smile effect." Plasmid fingerprinting can be utilized for epidemiologic studies of both nosocomial and community-acquired infections. The use of restriction endonuclease analysis can greatly enhance the ability of the investigator to differentiate strains that harbor only a single plasmid. Plasmid fingerprinting often provides the only differential characteristic for strains involved in epidemics.
Bacteria↗
Transpogenes: the transposition-like integration of short sequence DNA into the yeast 2 micron plasmid creates the STB locus and plasmid-size polymorphism.
The type-2 2 mu plasmid of industrial yeast strains exhibits extensive size polymorphism in the STB (plasmid stability) locus and IR (inverted repeat)-right region. Comparative DNA sequence analyses of STB alleles identified a 38-bp sequence flanked by a 25-bp direct repeat as the underlying structural motif. Variable unequal recombination within the direct repeat accounted for the observed polymorphism of STB alleles. IR-right polymorphism was observed to result from tandem duplication of a 22-bp sequence flanked by a 9-bp direct repeat. The flanking direct repeats marked both loci as originating from the transposition-like integration of short DNA fragments. We call these structures transpogenes and note that these are hybrid structures of host and foreign DNA which can evolve into functional loci.
Base Sequence↗
The sequences of the ribonucleotide reductase genes from African swine fever virus show considerable homology with those of the orthopoxvirus, vaccinia virus.
Two African swine fever virus (ASFV) recombinant plasmids containing large inserts of DNA have been sequenced at random, and translations of the DNA sequence have been compared to libraries of vaccinia virus protein sequences. Among other genes identified by their extensive homology with vaccinia virus genes were the large and small subunits of ribonucleotide reductase. A 5.5-kb fragment from the Malawi (LIL20/1) strain of ASFV was identified as containing the genes for both these subunits. The fragment has been sequenced and the two genes have been found to be in a head-to-head orientation. The sequences are compared to other sequenced ribonucleotide reductase genes, and the evolutionary implications discussed.
African Swine Fever Virus↗
Molecular evolution of hemagglutinin genes of H1N1 swine and human influenza A viruses.
The hemagglutinin (HA) genes of influenza type A (H1N1) viruses isolated from swine were cloned into plasmid vectors and their nucleotide sequences were determined. A phylogenetic tree for the HA genes of swine and human influenza viruses was constructed by the neighbor-joining method. It showed that the divergence between swine and human HA genes might have occurred around 1905. The estimated rates of synonymous (silent) substitutions for swine and human influenza viruses were almost the same. For both viruses, the rate of synonymous substitution was much higher than that of nonsynonymous (amino acid altering) substitution. It is the case even for only the antigenic sites of the HA. This feature is consistent with the neutral theory of molecular evolution. The rate of nonsynonymous substitution for human influenza viruses was three times the rate for swine influenza viruses. In particular, nonsynonymous substitutions at antigenic sites occurred less frequently in swine than in humans. The difference in the rate of nonsynonymous substitution between swine and human influenza viruses can be explained by the different degrees of functional constraint operating on the amino acid sequence of the HA in both hosts.
Amino Acid Sequence↗
Flashback to the 1960s: utility of archived sera to explore the origin and evolution of Plasmodium falciparum chloroquine resistance in the Pacific.
The increasing frequencies of Plasmodium falciparum strains that are resistant to chloroquine (CQ) and other antimalarials are resulting in a global resurgence of malaria morbidity and mortality. CQ resistance (CQR) is associated with multiple mutations in the P. falciparum chloroquine resistance transporter (pfcrt) gene. The mode and tempo of the accumulation of substitutions leading to these complex CQR haplotypes remain speculative due to the dearth of samples temporally spanning the evolution of drug resistance. The origin and evolution of the CQR alleles of Papua New Guinea (PNG) is particularly ambiguous. It remains unclear whether the pfcrt haplotype in PNG resulted from an independent origin of a CQR haplotype identical in sequence to the South American haplotype, or if this haplotype originated in South America and recombined into a Southeast Asian-derived genome. We sequenced a segment of pfcrt exon 2 from 398 plasmid clones derived from archival human sera collected in the Pacific before and after the first reported cases of CQ treatment failure (n=251) and modern samples (n=147). None of the 251 pfcrt plasmid clones from nine archival samples displayed the C72S or the K76T mutations that are characteristic of CQR strains. In contrast, these two amino acid substitutions were present in all 147 pfcrt plasmid clones from five samples collected between 2001 and 2003; thus, the archival samples represent the baseline parasite genetic diversity before the evolution of CQR strains. We are currently expanding our analyses to include additional samples from the series described here and from series collected in the 1970s and the 1980s to evaluate the geographic origin of CQR strains in the Pacific and the validity of the sequential point mutation accumulation model of CQR evolution.
Amino Acid Sequence↗
Evolution of hypervariable region 1 of hepatitis C virus in primary infection.
The hypervariable region 1 (HVR-1) of the putative envelope encoding E2 region of hepatitis C virus (HCV) RNA was analyzed in sequential samples from three patients with acute type C hepatitis infected from different sources to address (i) the dynamics of intrahost HCV variability during the primary infection and (ii) the role of host selective pressure in driving viral genetic evolution. HVR-1 sequences from 20 clones per each point in time were analyzed after amplification, cloning, and purification of plasmid DNA from single colonies of transformed cells. The intrasample evolutionary analysis (nonsynonymous mutations per nonsynonymous site [Ka], synonymous mutations per synonymous site [Ks], Ka/Ks ratio, and genetic distances [gd]) documented low gd in early samples (ranging from 2. 11 to 7.79%) and a further decrease after seroconversion (from 0 to 4.80%), suggesting that primary HCV infection is an oligoclonal event, and found different levels and dynamics of host pressure in the three cases. The intersample analysis (pairwise comparisons of intrapatient sequences; rKa, rKs, rKa/rKs ratio, and gd) confirmed the individual features of HCV genetic evolution in the three subjects and pointed to the relative contribution of either neutral evolution or selective forces in driving viral variability, documenting that adaptation of HCV for persistence in vivo follows different routes, probably representing the molecular counterpart of the viral fitness for individual environments.
Adolescent↗
Directed evolution of new glycosynthases from Agrobacterium beta-glucosidase: a general screen to detect enzymes for oligosaccharide synthesis.
BACKGROUND: Oligosaccharide synthesis is becoming increasingly important to industry as diverse therapeutic roles for these molecules are discovered. The chemical synthesis of oligosaccharides on an industrial scale is often prohibitively complex and costly. An alternative, that of enzymatic synthesis, is limited by the difficulty of obtaining an appropriate enzyme. A general screen for enzymes that catalyze the synthesis of the glycosidic bond would enable the identification and engineering of new or improved enzymes. RESULTS: Glycosynthases are nucleophile mutants of retaining glycosidases that efficiently catalyze the synthesis of the glycosidic linkage by condensing an activated glycosyl fluoride donor with a suitable acceptor sugar. A novel agar plate-based coupled-enzyme screen was developed (using a two-plasmid system) and used to select an improved glycosynthase from a library of mutants. CONCLUSIONS: Plate-based coupled-enzyme screens of this type are extremely valuable for identification of functional synthetic enzymes and can be applied to the evolution of a range of glycosyl transferases.
Directed Molecular Evolution↗