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A novel eIF2B-dependent mechanism of translational control in yeast as a response to fusel alcohols.

Fusel alcohols are natural products of amino acid catabolism in the yeast Saccharomyces cerevisiae that cause morphological changes similar to those seen during pseudohyphal growth. We have discovered that certain of these alcohols, including butanol and isoamyl alcohol, bring about a rapid inhibition of translation at the initiation step. This inhibition is strain specific and is not explained by previously described translational control pathways. Using genetic mapping, we have identified a proline to serine allelic variation at amino acid 180 of the GCD1 gene product as the genetic locus that allows translational regulation upon butanol addition. Gcd1p forms part of the eIF2B guanine nucleotide complex that is responsible for recycling eIF2-GDP to eIF2-GTP. This represents one of the key limiting steps of translation initiation and we provide evidence that fusel alcohols target eIF2B in order to bring about translational regulation.

Alcohols↗

Molecular evolution of the Escherichia coli chromosome. I. Analysis of structure and natural variation in a previously uncharacterized region between trp and tonB.

We present the sequence of a 3500-bp region of the Escherichia coli strain K12 chromosome lying between the tryptophan operon and the tonB gene. Analysis of the sequence yields six open reading frames that have properties characteristic of genes for proteins. The reading frames are closely spaced, and putative transcription units and control sites compose over 95% of the DNA. The sequences of several wild strains of E. coli have been determined for a large segment of the region described. Comparison of these sequences reveals the effects of base substitutions, DNA rearrangements, and recombination. In the regions presumably expressed as polypeptides, most of the natural variation results from synonymous substitutions. However, the DNA rearrangements identified have end points within the open reading frames and disrupt them in a variety of ways. The effects of genetic recombination between strains, recently found to be significant on a large scale in E. coli, are also apparent in the region between trp and tonB.

Base Sequence↗

The sequence of the nucleoprotein gene of human influenza A virus, strain A/NT/60/68.

The nucleotide sequence of the nucleoprotein gene of influenza A/NT/60/68 was established after using improved cloning methods to obtain full length cDNA clones in pBr322. The gene is 1565 residues long and codes for a basic protein of 498 amino acids. There are only 30 amino acid differences between it and the homologous sequence in A/PR/8/35, all occurring as point mutations. Assuming a common lineage, the evolutionary rate of divergence of the two strains is 0.18% amino acid per year. This confirms there is a slow but significant rate of evolution.

Amino Acid Sequence↗

Nucleotide sequence of the Escherichia coli gap gene. Different evolutionary behavior of the NAD+-binding domain and of the catalytic domain of D-glyceraldehyde-3-phosphate dehydrogenase.

A 1523-base-pair DNA fragment, spanning the gap gene from Escherichia coli, has been sequenced. It contains an open-reading frame whose length (330 amino acids) is in agreement with D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) molecular mass. This coding sequence is preceded by a Shine-Dalgarno complementary sequence and by two overlapping promoter-like structures. The codon usage within gap is consistent with that expected for a gene which is strongly expressed. The amino acid sequence of the E. coli GAPDH, deduced from the DNA sequence, contains all the amino acids postulated to play a functional role in GAPDH. Comparison of the E. coli enzyme with enzymes from other species reveals different evolutionary behaviour of the NAD+-binding domain and of the catalytic domain of GAPDH. The E. coli enzyme is found to be more similar to eucaryotic enzymes than to enzymes from thermophilic bacteria. This observation is discussed in terms of adaptation to growth at high temperature.

Amino Acid Sequence↗

The Arms Race Between Actinobacillus pleuropneumoniae and Its Genetic Environment: A Comprehensive Analysis of Its Defensome and Mobile Genetic Elements.

Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia in swine, a highly contagious and economically significant disease. The genetic variability of A. pleuropneumoniae complicates disease control efforts, as it enables rapid adaptation to various stressors, including antimicrobial treatments. To better understand the molecular mechanisms underlying this adaptability, we investigated the role of the bacterial defensome and its relationship with mobile genetic elements (MGEs), such as prophages, plasmids, and integrative conjugative elements (ICEs). Using bioinformatic tools, we identified a diverse and rich defensome in A. pleuropneumoniae, with an average of 16 different defense systems per strain. We found that CRISPR-Cas systems, along with other defense mechanisms, are actively involved in restricting the entry of foreign genetic material, playing a crucial role in bacterial adaptation. Additionally, we characterized several novel prophages and examined their distribution across different strains, revealing their potential contribution to the bacterium's evolutionary success. Our findings underscore the complex interplay between the bacterium's defense systems and MGEs, shedding light on how A. pleuropneumoniae maintains genetic diversity while also safeguarding itself against external threats. These insights provide a better understanding of the genetic factors that influence the pathogen's adaptability and highlight potential avenues for more effective disease control strategies.

Actinobacillus pleuropneumoniae↗

The composite genome of the legume symbiont Sinorhizobium meliloti.

The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.

Bacterial Adhesion↗

Evolution of TEM-related extended-spectrum beta-lactamases in Korea.

TEM-52, differing from TEM-1 by having the substitutions Glu-104-->Lys, Met-182-->Thr, and Gly-238-->Ser, has previously been described as the most prevalent extended-spectrum beta-lactamase (ESBL) in Korea. In a further survey, we discovered the ESBLs TEM-15, which is like TEM-52 but lacks the substitution at residue 182, and TEM-88, which is like TEM-52 with an additional Gly-196-->Asp substitution. TEM-88 retained the activity of TEM-52 against moxalactam. Otherwise, the kinetic properties of the three ESBLs failed to show an advantage to this evolution.

Anti-Bacterial Agents↗

Sequence of the Streptococcus pneumoniae bacteriophage HB-3 amidase reveals high homology with the major host autolysin.

We have sequenced a DNA fragment containing the pneumococcal bacteriophage HB-3 hbl gene, which codes for the phage lytic amidase. A remarkable nucleotide similarity (87.1%) between the lytA gene, coding for the pneumococcal amidase, the major autolysin of Streptococcus pneumoniae, and the hbl gene was found. This similarity completely disappeared outside the open reading frames coding for both amidases. The hbl gene transformed amidase-deficient strains of S. pneumoniae to the wild-type phenotype, and Southern blotting experiments provided evidence for recombination between donor and recipient genes. A comprehensive evaluation of these and previous results on the peptidoglycan hydrolases of S. pneumoniae and its bacteriophages suggested that recombination mechanisms participate in the evolution of the genes coding for these enzymes.

Amidohydrolases↗

Genes coding for the selenocysteine-inserting tRNA species from Desulfomicrobium baculatum and Clostridium thermoaceticum: structural and evolutionary implications.

The genes (selC) coding for the selenocysteine-inserting tRNA species (tRNA(Sec)) from Clostridium thermoaceticum and Desulfomicrobium baculatum were cloned and sequenced. Although they differ in numerous positions from the sequence of the Escherichia coli selC gene, they were able to complement the selC lesion of an E. coli mutant and to promote selenoprotein formation in the heterologous host. The tRNA(Sec) species from both organisms possess all of the unique primary, secondary, and tertiary structural features exhibited by E. coli tRNA(Sec) (C. Baron, E. Westhof, A. Böck, and R. Giegé, J. Mol. Biol. 231:274-292, 1993). The structural and functional properties of the tRNA(Sec) species from prokaryotes analyzed thus far support the notion that tRNA(Sec) may be an evolutionarily conserved structure whose function in the primordial genetic code was to decode UGA with selenocysteine.

Base Sequence↗

Directed evolution of retrovirus envelope protein cytoplasmic tails guided by functional incorporation into lentivirus particles.

In contrast to most gammaretrovirus envelope proteins (Env), the Gibbon ape leukemia virus (GaLV) Env protein does not mediate the infectivity of human immunodeficiency virus type 1 (HIV-1) particles. We made use of this observation to set up a directed evolution system by creating a library of GaLV Env variants diversified at three critical amino acids, all located around the R-peptide cleavage site within the cytoplasmic tail. This library was screened for variants that were able to functionally pseudotype HIV-1 vector particles. All selected Env variants mediated the infectivity of HIV-1 vector particles and encoded novel cytoplasmic tail motifs. They were efficiently incorporated into HIV particles, and the R peptide was processed by the HIV protease. Interestingly, in some of the selected variants, the R-peptide cleavage site had shifted closer to the C terminus. These data demonstrate a valuable approach for the engineering of chimeric viruses and vector particles.

Amino Acid Sequence↗

Detection of HIV-2 by PCR.

Genomes of human immunodeficiency virus type 2 (HIV-2), like those of HIV-1 or other retroviruses, are highly variable. These genetic variants have been classified into seven genetic subtypes. The average genetic divergence between different subtypes is about 20% in the gag gene, which is higher than those among HIV-1 group M subtypes. The current serological tests cannot distinguish the different subtypes from one another. To understand genetic variation, evolution, and subtype distribution of HIV-2, polymerase chain reaction (PCR) technology has been widely used. The PCR products that are amplified from highly conserved regions among all subtypes can be either cloned into plasmid vectors for sequence analysis or directly sequenced without a cloning step. Phylogenetic analysis of newly obtained sequences with reference sequences can determine the subtype classification or identify new subtypes if the sequences do not belong to any known subtypes.

DNA, Viral↗

Sequence-specific interaction between S1F, a spinach nuclear factor, and a negative cis-element conserved in plastid-related genes.

The nuclear gene rps1 coding for the spinach plastid ribosomal protein CS1 exhibits both a constitutive and leaf-specific expression pattern. In contrast to other chloroplast-related genes like rbcS and cab, the leaf induction of rps1 expression is light-independent. These unique features of rps1 expression provide good models to study the mechanisms regulating plastid development and differentiation in higher plants. We report on the identification of a spinach leaf nuclear factor, designated S1F, interacting with the rps1 promoter. The S1F binding site is conserved in the promoter region of many plastid-related genes, including rbcS, cab, and rpl21. A binding activity similar to S1F was detected in nuclear extract from dark-grown de-differentiated soybean suspension cells. Through site-specific mutagenesis and transient expression in soybean cell protoplasts, we show that the S1F binding site is a negative element down-regulating the promoter activity of rps1. A ligated tetramer of S1F site was able to repress activity of the cauliflower mosaic virus 35 S promoter extending the negative function of the S1F binding site on promoter activity.

Base Sequence↗

[Horizontal gene transfer].

In this paper the conception of horizontal gene transfer (HGT) was introduced,and main mode of HGT was also enumerated as follows: HGT by medium such as plasmid and virus etc., and the HGT without any medium. The transfer of genes from one species to another especially between remote species was emphasized by the information from genome sequencing. The problems about evolution phylogenies and safety of GEMs (gene engineered microorganisms) for HGT were discussed.

English Abstract↗

[Variability analysis of S2 gene of SARS-CoV].

OBJECTIVE: To determine the sequence of S2 gene of SARS-associated coronavirus (SARS-CoV) GD322 and analyze the phyletic evolution of S2 gene. METHOD: S2 gene fragment was amplified from SARS-CoV GD322 genome with RT-PCR and ligated to pGEM-T vector for sequence analysis after transformation of the plasmid into E. coli DH5a. The variability of S2 genes and S2 proteins from 12 strains isolated in the early, intermediate and advanced stages of the SARS outbreak were analyzed and the phylogenetic tree was constructed with Lasergene, Clustal X, DNAman and Treeview. T cell antigen epitopes of S2 protein were predicted on the basis of Internet database. RESULT: With the epidemic spread of SARS-CoV, the S2 genes of the virus tended to become stable. Homology of S2 genes of SARS-CoV isolated in advanced stage of the outbreak reached 99.9%. Prediction of T cell antigen epitope showed that mutation at the 57th amino acid effected T cell antigen epitope. CONCLUSION: S2 gene of GD322 SARS-CoV is relatively stable during the epidemic spread of the virus, and mutation at the 57th amino acids of S2 protein may affect the T cell antigen epitope.

Escherichia coli↗

Molecular structures of human argininosuccinate synthetase pseudogenes. Evolutionary and mechanistic implications.

In the human genome there is one expressed gene for argininosuccinate synthetase and 14 pseudogenes. A cDNA coding for human argininosuccinate synthetase was used to screen a human genomic library. Twenty-five unique genomic clones were isolated and extensively characterized. At least seven clones represented processed argininosuccinate synthetase pseudogenes that lost the introns in the expressed gene. Restriction mapping demonstrated that these processed pseudogenes were located in distinct regions of the human genome. Complete nucleotide sequences of two processed pseudogenes, psi AS-1 and psi AS-3, and a partial sequence of psi AS-7 were determined. Both psi AS-1 and psi AS-3 had an adenine-rich region at their 3' end and were flanked by distinct imperfect direct repeats. A comparison of these pseudogene sequences to that of the cDNA demonstrated that psi AS-1 and psi AS-3 were 93% homologous to the cDNA, whereas psi AS-7 was 89% homologous to the cDNA. Therefore, it is estimated that psi AS-1 and psi AS-3 were created 10-11 million years ago, whereas psi AS-7 arose approximately 21 million years ago. We have estimated the evolutionary rate for the expressed argininosuccinate synthetase gene based on the sequences of psi AS-1 and psi AS-3. These data indicate that the expressed argininosuccinate synthetase gene is evolving at a rate similar to that of the beta-globin gene and much faster than the alpha-tubulin gene. Furthermore, a comparison of the sequences of psi AS-1 and psi AS-3 suggests the possibility that these pseudogenes arose from a common intermediate.

Argininosuccinate Synthase↗

The nucleotide sequence, expression, and evolution of one member of a multigene family encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in soybean.

We have examined the nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate (RuBP) carboxylase from soybean. One member of this gene family, designated SRS1, has been isolated from a soybean DNA library constructed in the lambda vector Charon 4A. The complete nucleotide sequence and structure of this gene including its two introns and portions of the 5' and 3' flanking sequences were determined. The first exon encodes the entire transit peptide (55 amino acids) and the first 2 amino acids of the mature sequence. Based on analysis of the nucleotide sequence, we concluded that the precursor of the soybean small subunit consists of 178 amino acids. A gene-specific probe for SRS1 was used to show that this gene is transcribed and that steady-state levels of its transcript are strongly light regulated. S1 nuclease mapping was used to locate the potential start of transcription in the sequence and showed that the small subunit gene contains a cap site, TATA box, and -80 sequence, which match the consensus animal sequences. The mature SRS1 small subunit polypeptide of 123 amino acids contains 30 and 34 amino acid replacements relative to the previously determined amino acid sequences from pea and spinach, respectively. Southern blotting of restriction digests of soybean nuclear DNA and data on the complete structure of SRS1 suggest that a multigene family of at least 10 members encodes the RuBP carboxylase small subunit in soybean. Quantitative evolutionary comparison of the soybean small subunit sequence for SRS1 and the pea small subunit sequence suggests that these two genes diverged long before the divergence of pea and soybean.

Amino Acid Sequence↗