A metabolic prototype for eliminating tryptophan from the genetic code.

We set out to reduce the chemical constitution of a living organism to 19 amino acids. A strain was constructed for reassigning the tryptophan codon UGG to histidine and eliminating tryptophan from Escherichia coli. Histidine codons in the gene for an essential enzyme were replaced with tryptophan codons and the restoration of catalytic activity by missense suppressor His-tRNA bearing a CCA anticodon was selected. We used automated cultivation to assess the stability of this genetic construct during evolution. Histidine to tryptophan mutation at codon 30 in the transketolase gene from yeast and its cognate suppressor tRNA were stably propagated in a tktAB deletant of E. coli over 2500 generations. The ratio of histidine misincorporation at tryptophan sites in the proteome increased from 0.0007 to 0.03 over 300 days of continuous culture. This result demonstrated that the genetic code can be forced to evolve by permanent metabolic selection.

New pseudo-stationary phases for electrokinetic capillary chromatography. Complexes between bovine serum albumin and sodium dodecyl sulfate.

The complexes formed between a protein (bovine serum albumin, BSA) and a surfactant (sodium dodecyl sulfate, SDS) were studied as separation carriers in electrokinetic chromatography. Selectivities different from those with either SDS or BSA alone in the background electrolyte (BGE) were obtained. Separation performances were demonstrated to be closely related to the type of complex formed, as predicted by the isotherm curve of SDS on BSA. For each composition of background electrolyte, capacity factors and resolutions were calculated. We compared the results with these complexes to electropherograms using BGE containing either BSA or SDS alone. The separation of a mixture of phenols indicate that some compositions of the BSA-SDS complexes are efficient selectors.

Counterion Effects in Aqueous Solutions of Cationic Surfactants: Electromotive Force Measurements and Thermodynamic Model.

EMF measurements have been performed to study the influence of counterions in aqueous solutions of ionic surfactants in which the amphiphile is an n-alkyltrimethylammonium cation. General rules of variation were observed for the activities of the amphiphilic ion and the counterion, as a function of the global concentration of the surfactant salt. This study also allows us to have access to the degree of counterion binding beta over a wide range of counterions (bromide, chloride, fluoride, hydroxide, nitrate, acetate, benzoate). A systematic correlation between the concentrations of an amphiphilic cation and a counterion in solution has been established when the micellization process occurs. This correlation is also verified when a background electrolyte with a common anion is added to the surfactant solution. The mathematical relation translating the real link between concentrations of both ions of the micellar salt, deduced from this correlation, emphasizes the existence of a condition of micellization, in the form of a micellization product. A new thermodynamic model based on the electrochemical equilibrium of a dispersed phase (pseudo-phase) is developed, and we justify the existence of this condition of micellization or micellization product. Furthermore, this model is shown to be well adapted for the description of the whole phenomena observed in micellar solutions. Copyright 1999 Academic Press.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XV.

Chromosome XV was one of the last two chromosomes of Saccharomyces cerevisiae to be discovered. It is the third-largest yeast chromosome after chromosomes XII and IV, and is very similar in size to chromosome VII. It alone represents 9% of the yeast genome (8% if ribosomal DNA is included). When systematic sequencing of chromosome XV was started, 93 genes or markers were identified, and most of them were mapped. However, very little else was known about chromosome XV which, in contrast to shorter chromosomes, had not been the object of comprehensive genetic or molecular analysis. It was therefore decided to start sequencing chromosome XV only in the third phase of the European Yeast Genome Sequencing Programme, after experience was gained on chromosomes III, XI and II. The sequence of chromosome XV has been determined from a set of partly overlapping cosmid clones derived from a unique yeast strain, and physically mapped at 3.3-kilobase resolution before sequencing. As well as numerous new open reading frames (ORFs) and genes encoding tRNA or small RNA molecules, the sequence of 1,091,283 base pairs confirms the high proportion of orphan genes and reveals a number of ancestral and successive duplications with other yeast chromosomes.

Sequence and analysis of a 26.9 kb fragment from chromosome XV of the yeast Saccharomyces cerevisiae.

We have determined the nucleotide sequence of a fragment of chromosome XV of Saccharomyces cerevisiae cloned into cosmid pEOA048. The analysis of the 26,857 bp sequence reveals the presence of 19 open reading frames (ORFs), and of one RNA-coding gene (SNR17A). Six ORFs correspond to previously known genes (MKK1/SSP32, YGE1/GRPE/MGE1, KIN4/KIN31/KIN3, RPL37B, DFR1 and HES1, respectively), all others were discovered in this work. Only five of the new ORFs have significant homologs in public databases, the remaining eight correspond to orphans (two of them are questionable). O5248 is a probable folypolyglutamate synthetase, having two structural homologs already sequenced in the yeast genome. O5273 shows homology with a yeast protein required for vanadate resistance. O5268 shows homology with putative oxidoreductases of different organisms. O5257 shows homology with the SAS2 protein and another hypothetical protein from yeast. The last one, O5245, shows homology with a putative protein of Caenorhabditis elegans of unknown function. The present sequence corresponds to coordinates 772,331 to 799,187 of the entire chromosome XV sequence which can be retrieved by anonymous ftp (ftp. mips. embnet. org).

Construction of a complete genomic library of Saccharomyces cerevisiae and physical mapping of chromosome XI at 3.7 kb resolution.

A consortium of European laboratories has been organized to systematically sequence the genome of Saccharomyces cerevisiae. As part of the BIOTECH program aimed at sequencing chromosomes XI and II, we have constructed a total genomic library of yeast strain FY1679 (a direct S288C derivative) into cosmid vectors pWE15 and pOU61cos. Primary clones from four independent libraries totalling 190 genome equivalents have been stored at -80 degrees C. A subset of 1939 independent clones (six genome equivalents) was hybridized using purified chromosomes XI and X as probes. A total of 147 chromosome XI-specific cosmid clones was used to construct the physical map of that chromosome. Mapping methods included a combination of classical bottom-up strategies (fingerprinting, hybridizations) and a novel top-down strategy using I-SceI chromosome fragmentation. The 147 cosmid clones form a unique contig covering the entire chromosome XI (666 kb) with the sole exceptions of the (C1-3A)n repeats of the telomeres. Colinearity of cosmid inserts with yeast DNA was directly verified. A complete EcoRI map of chromosome XI was deduced from partial overlaps of cosmids and used for the sequencing program. Comparison of this map with the genetic map shows unexpected divergences that have been solved by subsequent genetic analysis, yet underline the necessity of independent physical mapping in genome projects.

Complete DNA sequence of yeast chromosome XI.

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

A 12.8 kb segment, on the right arm of chromosome II from Saccharomyces cerevisiae including part of the DUR1,2 gene, contains five putative new genes.

A 12,820 bp fragment from the right arm of chromosome II of Saccharomyces cerevisiae was sequenced and analysed. This fragment contains six non-overlapping long open reading frames (ORFs) designated from the centromere- to the telomere-proximal ends as: YBR1441, 1443, 1444, 1445, 1446 and 1448. YBR1441 encodes a polypeptide of 845 amino acids which shares a long consensus domain with products of S. cerevisiae MCM2, MCM3, CDC46 and Schizosaccharomyces pombe cdc21+ genes. These genes are involved in DNA replication. YBR1445 encodes a polypeptide of 404 amino acids which has strong similarity with the S. cerevisiae KRE2/MNT1, YUR1, KTR1 gene products. The KRE2/MNT1 protein is an alpha-1,2- mannosyltransferase. The product of YBR1444, which encodes a protein of 375 amino acids, presents a lipase signature sequence and a peroxisomal targeting signal. YBR1448, whose sequence extends further on the telomere-proximal end of the fragment, is identical to the 3' end of the DUR1,2 gene encoding urea amidolyase. The two ORFs, YBR1443 and YBR1446, exhibit no significant similarity with any known gene.