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At least 523 records · Page 29Linked to original sources
PubMed1994

The roles of starvation and selective substrates in the emergence of araB-lacZ fusion clones.

The araB-lacZ fusion system has been a key case in the 'directed mutation' controversy. Fusions did not occur detectably during normal growth but formed readily after prolonged incubation on selective Ara-Lac medium. To distinguish the roles of starvation stress and selective substrates in coding sequence fusions, we applied sib selection and PCR technologies. Sib selection of the prefusion strain, MCS2, starved under aerobic conditions permitted us to isolate active fusion clones which had never been in contact with arabinose or lactose. Hence, a directive role for selective substrates is not essential. Aerobiosis was necessary for fusions to appear in glucose-starved cultures. The difference in fusion formation between normal and starved conditions is best explained by the response of a signal transduction network to physiological stimuli to activate Mu prophage joining of araB and lacZ sequences. PCR analysis revealed that direct plating on selective Ara-Lac agar yielded mostly a single class of 'standard' fusions, while sib selection yielded a broader spectrum of fusion structures. Standard fusions were found to occur within a narrow 9 bp window in lacZ. The high frequency of standard fusions in glucose-starved cultures suggested efficient and/or specific Mu action.

Base Sequence↗
PubMed1999

The relationship between protein structure and function: a comprehensive survey with application to the yeast genome.

For most proteins in the genome databases, function is predicted via sequence comparison. In spite of the popularity of this approach, the extent to which it can be reliably applied is unknown. We address this issue by systematically investigating the relationship between protein function and structure. We focus initially on enzymes functionally classified by the Enzyme Commission (EC) and relate these to by structurally classified domains the SCOP database. We find that the major SCOP fold classes have different propensities to carry out certain broad categories of functions. For instance, alpha/beta folds are disproportionately associated with enzymes, especially transferases and hydrolases, and all-alpha and small folds with non-enzymes, while alpha+beta folds have an equal tendency either way. These observations for the database overall are largely true for specific genomes. We focus, in particular, on yeast, analyzing it with many classifications in addition to SCOP and EC (i.e. COGs, CATH, MIPS), and find clear tendencies for fold-function association, across a broad spectrum of functions. Analysis with the COGs scheme also suggests that the functions of the most ancient proteins are more evenly distributed among different structural classes than those of more modern ones. For the database overall, we identify the most versatile functions, i.e. those that are associated with the most folds, and the most versatile folds, associated with the most functions. The two most versatile enzymatic functions (hydro-lyases and O-glycosyl glucosidases) are associated with seven folds each. The five most versatile folds (TIM-barrel, Rossmann, ferredoxin, alpha-beta hydrolase, and P-loop NTP hydrolase) are all mixed alpha-beta structures. They stand out as generic scaffolds, accommodating from six to as many as 16 functions (for the exceptional TIM-barrel). At the conclusion of our analysis we are able to construct a graph giving the chance that a functional annotation can be reliably transferred at different degrees of sequence and structural similarity. Supplemental information is available from http://bioinfo.mbb.yale.edu/genome/foldfunc++ +.

Enzymes↗
PubMed2000

Split gene origin and periodic introns.

The introns-early view has been challenged for several genes; prominent instances are triose phosphate isomerase (TPI), aldolase, pyruvate kinase (PK), alcohol dehydrogenase (ADH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and myosin heavy chain. While some of their introns appear to be phylogenetically ancient and/or to delineate exons corresponding to protein modules, a considerable number seemingly do not. But it is argued here that many of these anomalous introns are periodic, that is, relics of internal sequence repetitions within the ancestral gene. Some of these periodic-intron patterns are shared between related genes, as in the alphabeta -barrels of TPI, aldolase and PK, or the Rossmann nucleotide-binding domain common to PK, ADH and GAPDH. This is further evidence for the ancestral status of these introns. The myosin heavy chain C-terminal rod region is paradoxical in that its sequence is clearly periodic but its intron placements are not; however, they exhibit a remarkable coherence of intron translational phases, suggesting that these introns may also have originally had a periodic arrangement now obscured by intron slipping.

Alcohol Dehydrogenase↗
PubMed1991

The synthesis of the two S-adenosyl-methionine synthetases is differently regulated in Saccharomyces cerevisiae.

S-adenosyl-L-methionine (AdoMet) is synthesized by transfer of the adenosyl moiety of ATP to the sulfur atom of methionine. This reaction is catalysed by AdoMet synthetase. In all eukaryotic organisms studied so far, multiple forms of AdoMet synthetases have been reported and from their recent study, it appears that AdoMet synthetase is an exceptionally well conserved enzyme through evolution. In Saccharomyces cerevisiae, we have demonstrated the existence of two AdoMet synthetases encoded by genes SAM1 and SAM2. Yeast, which is able to concentrate exogenously added AdoMet, is thus a particularly useful biological system to understand the role and the physiological significance of the preservation of two almost identical AdoMet synthetases. The analysis of the expression of the two SAM genes in different genetic backgrounds during growth under different conditions shows that the expression of SAM1 and SAM2 is regulated differently. The regulation of SAM1 expression is identical to that of other genes implicated in AdoMet metabolism, whereas SAM2 shows a specific pattern of regulation. A careful analysis of the expression of the two genes and of the variations in the methionine and AdoMet intracellular pools during the growth of different strains lead us to postulate the existence of two different AdoMet pools, each one supplied by a different AdoMet synthetase but in equilibrium with each other. This could be a means of storing AdoMet whenever this metabolite is overproduced, thus avoiding the degradation of a metabolite the synthesis of which is energetically expensive.

Blotting, Northern↗
PubMed1977

Genic homozygosity in an ancient reptile (Alligator mississippiensis).

Proteins assayed electrophoretically showed variation at only three of 49 presumed genetic loci in alligators from southwestern Louisiana. Average heterozygosity per individual was 0.021+/-0.012; proportion of polymorphic loci was 0.06. Data on the history, structure, and ecology of this alligator population are consistent with natural selection as the primary factor accounting for this low genetic variability. However, neither a historic population bottleneck nor some genetic mechanism limiting variability can be dismissed as a possible factor.

Alligators and Crocodiles↗
PubMed1984

Biochemical diversity and evolution in the genus Mus.

Thirteen biochemical groups of wild mice from Europe, Asia, and Africa belonging to the genus Mus are analyzed at 22-42 protein loci. Phylogenetic trees are proposed and patterns of biochemical evolution are discussed, as well as the possible contribution of wild mice to the genetic diversity of laboratory stocks.

Alleles↗
PubMed1996

Identification of cyclized calmodulin antagonists from a phage display random peptide library.

To isolate peptide ligands that bound calmodulin (CaM) specifically, we screened an M13 phage library displaying cyclized octamer random peptides with immobilized bovine CaM. Isolates were recovered, sequenced, and deduced to express nine independent peptides, five of which contained the sequence Trp-Gly-Lys (WGK). Four of the nine peptide sequences were synthesized in cyclized, biotinylated form. All of the peptides required Ca2+ to bind CaM. The cyclized, disulfide-bonded form of one such peptide, SCLRWGKWSNCGS, bound CaM better than its reduced form or an analogue in which the cysteine residues were replaced by serine. The cyclized peptide also exhibited the ability to inhibit CaM-dependent kinase activity. Systematic alanine substitution of residues in this peptide sequence implicate the tryptophan residue as being critical for binding, with other residues contributing to binding to varying degrees. Cloning of ligand targets (COLT) confirmed the specificity of one of the cyclized peptides, yielding full-length and C-terminal CaM clones, in addition to a full-length clone of troponin C, a CaM-related protein. This study has demonstrated that conformationally constrained peptides isolated from a phage library acted as specific, Ca(2+)-dependent CaM ligands.

Amino Acid Sequence↗
PubMed1977

Evolutionary adaptability of biological macromolecules.

The degree of gradualism with which tertiary structure and function of protein changes with stepwise changes in primary structure (assumed to be influenced by redundancy of weak bonding) is both a precondition for and consequence of evolution. The resulting selection for degree of gradualism has implications for a number of structural and functional properties of modern proteins as well as for the significance of neutral (so-called non-Darwinian) phenomena in relation to selection.

Amino Acids↗