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In rapidly adapting asexual populations, including many microbial pathogens and viruses, numerous mutant lineages often compete for dominance within the population.sup.1-5. These complex evolutionary dynamics determine the outcomes of adaptation, but have been difficult to observe directly. Previous studies have used whole-genome sequencing to follow molecular adaptation.sup.6-10; however, these methods have limited resolution in microbial populations. Here we introduce a renewable barcoding system to observe evolutionary dynamics at high resolution in laboratory budding yeast. We find nested patterns of interference and hitchhiking even at low frequencies. These events are driven by the continuous appearance of new mutations that modify the fates of existing lineages before they reach substantial frequencies. We observe how the distribution of fitness within the population changes over time, and find a travelling wave of adaptation that has been predicted by theory.sup.11-17. We show that clonal competition creates a dynamical 'rich-get-richer' effect: fitness advantages that are acquired early in evolution drive clonal expansions, which increase the chances of acquiring future mutations. However, less-fit lineages also routinely leapfrog over strains of higher fitness. Our results demonstrate that this combination of factors, which is not accounted for in existing models of evolutionary dynamics, is critical in determining the rate, predictability and molecular basis of adaptation. A renewable barcoding system reveals the evolutionary dynamics of laboratory budding yeast, showing that fitness changes over time in a travelling wave of adaptation that can fluctuate owing to leapfrogging events., Author(s): Alex N. Nguyen Ba [sup.1] , Ivana Cvijovic [sup.1] [sup.2] [sup.3] [sup.4] , José I. Rojas Echenique [sup.1] , Katherine R. Lawrence [sup.1] [sup.5] , Artur Rego-Costa [sup.1] , [...]

Defining the transcriptomic identity of malignant cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To address this challenge, here we developed Genotyping of Transcriptomes (GoT), a method to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. We apply GoT to profile 38,290 CD34.sup.+ cells from patients with CALR-mutated myeloproliferative neoplasms to study how somatic mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant versus normal haematopoietic progenitors revealed an increasing fitness advantage with myeloid differentiation of cells with mutated CALR. We identified the unfolded protein response as a predominant outcome of CALR mutations, with a considerable dependency on cell identity, as well as upregulation of the NF-[kappa]B pathway specifically in uncommitted stem cells. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in myeloproliferative neoplasms is dependent on the native cell identity. Profiling of over 38,000 CD34.sup.+ cells from patients with CALR-mutated myeloproliferative neoplasms, using the 'Genotyping of Transcriptomes' procedure, reveals that the transcriptional output of these mutations depends upon native cell identity., Author(s): Anna S. Nam [sup.1] [sup.2] [sup.3] , Kyu-Tae Kim [sup.2] [sup.3] [sup.4] , Ronan Chaligne [sup.2] [sup.3] [sup.4] , Franco Izzo [sup.2] [sup.3] [sup.4] , Chelston Ang [sup.2] [sup.3] [...]

A method for detecting mutations and measuring gene-expression levels in the same cell has enabled an investigation into the effects of mutations in a specific gene on the emergence of a form of blood cancer. A combined method probes DNA and RNA in blood progenitor cells., Author(s): Siddharth Raju, Chun Jimmie Ye Author Affiliations: How mutations express themselves in blood-cell production The cells that circulate in the bloodstream perform various functions and, in adults, are derived [...]

BACKGROUND: 1,3-Butadiene (BD) is an important industrial chemical and an environmental and occupational pollutant. The carcinogenicity of BD in rodents has been proved, but its carcinogenic and mutagenic molecular mechanism(s) [...]

A hybrid quantum-classical molecular dynamics simulation of a mutant escherichia coli dihydrofolate reductase enzyme is presented. The simulations suggest that this mutation disturbs a network of coupled promoting motions, which is important in the dihydrofolate reductase catalysis.

The mutations of R248 to Trp and Gln in the core domain (CD) of the p53 protein are some of the most common mutations found in human cancer. Although the mutant 248Q and 248W p53-CDs retain the wild-type conformation and stability, they lack sequence-specific DNA binding and transactivation functions and the ability to suppress cell growth.

Cyclic ichthyosis with epidermolytic hyperkeratosis is a phenotype that results from mutations in the keratin K1 2B domain. Four persons from two families with a unique clinical disorder with histological findings of epidermolytic hyperkeratosis are described. At birth they manifested erythema and superficial erosions. They improved during the next few months and later developed palmoplantar hyperkeratosis with scale and patchy erythema. A clinical phenotype different from classic bullous congential ichthyosiform erythroderma (BCIE), but with similar histology, can be the result of K1 mutations.

The hypothesis that humans may suffer a high genomic deleterious mutation rate has been tested through application of a variant of a molecular approach. It is estimated that around 4.2 amino-acid-altering mutations per diploid per generation have occurred since separation from chimpanzees. About 38% are estimated to have been eliminated by natural selection, suggesting over 1.6 new deleterious mutations, and indicating that the rate specific to protein-coding sequences is nearly to the upper tolerable limit.

Mutations that generate premature-termination codons (PTCs) may bring on exon-skipping by a number of mechanisms. Stop mutations disrupt gene functions in different ways. They give rise to truncated polypeptides because of the PTCs and often affect metabolism of corresponding mRNAs. In neurofibromin transcripts from different neurofibromatosis type 1 (NF1) patients have skipping of exons that have PTCs. Several stop mutations in some base pairs in exons 7 and 37 have been characterized. Creation/disruption of exon sequences related to splicing and changes in the secondary structure of mRNA may cause splicing events seen in the NF1 gene.

A study of two meristic mutations in Phacelia dubia (L.) Trel. shows that one causes the development of one additional sepal, petal and stamen, while the other produces triseriate pistil with trifurcating style and a tricarpellate ovary. The ovary has three functional placentae and show tripartite dehiscence. This gynoecium phenotype is controlled by recessive alleles located at two nuclear encoded loci. The gynoecium phenotype is similar to that found in Polemoniaceae, which indicates that simple genetic variation is involved in the variation of gynoecium phenotype in different families.

Mass extinctions result from unsuccessful mutations which are caused by atomic radiations coming from cosmos. This process is dependent on the galactic component of cosmic rays. The radiations generate new species which finally evolve into higher forms. The radiations increase to high levels after crossings of the galactic plane by the sun leading to mass extinctions. When the level of radiation falls back to normal, life continues with different flora and fauna.

A mutation at Phe(super 19) of pigeon malic enzyme fusion protein is responsible for the reduced activity of the triple F19S/N250S/L353Q mutant. The substitution of Phe(super 19) by nonaromatic residues reduces the k(sub cat) value, and the Mn2+ and L-malate binding affinities. This indicates that the Phe(super 19) is present in or near the Mn2+ -L-malate binding site, and the active region for subunit interaction.

Tryptophan (Trp) -containing mutants of the Escherichia coli transport protein, enzyme II(super mtl) (EII(super mtl)), are structurally similar to the wild-type protein. EII(super mtl) is involved in mannitol transport, and the mutants are able to bind, transport, and phosphorylate mannitol. The fluorescent spectra shows that the Trp at the 384th position is highly polar, that at the 42nd, 109th and 117th position is polar, while that at the 30th position is apolar. The binding of mannitol to Trp at the 30th position increases the fluorescence of the wild-type enzyme.

The replacement of 184 methionine of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) with valine at codon 184 adds to the fidelity and efficiency of DNA synthesis. M184 is the second residue of the conserved YXDD motif, located in RNA dependent DNA polymerases. The mutants M184A and M184V in which the Met residue is replaced with Ala or Val are catalytically more efficient than wild type enzyme. A three dimensional molecular model of HIV-1 RT ternary complex reveals that the position of M184 permits interaction with sugar motif of the primer nucleotide or dNTP substrate.

Electron and optical paramagnetic resonance spectroscopic analyses of the binding of exogenous ligands to the Met121X mutant of Pseudomonas aeruginosa azurin indicate that small molecules bind to copper through the cavity formed by the side chain of Met121. The strong absorption bands of azurin develop due to S-to-Cu charge transfer transitions. Small carboxylic acids react strongly with metal ions and copper attains a type 1.5 character. The EPR spectra of azurin are discussed.

The modulation of ionic interactions has no effect on the binding of phosphate to its active transport receptor in a phosphate binding protein from Escherichia coli. Local induced dipoles and hydrogen bonds stabilize the charge on the phosphate and compensate for the damage done by modulation of ionic interactions. The mutations in Arg 135 and Asp 137 in the protein using site directed mutagenesis induce modulations of ionic interactions.

Genetic and sequence analysis indicates that the His212, His268, and Asp214 endogenous ligands form the ferrous active site of isopenicillin N synthase (IPNS) from Streptomyces jumonjinensis. Mutations in these residues remove enzymatic activity. The fourth ligand is a glutamine residue that is probably replaced when the substrate binds to the enzyme. Enzymatic activity is unaffected by site directed mutagenesis of the other histidine residues and aspartic acid residues which are conserved in bacterial and fungal IPNS. These substitutions produce no observable conformational changes.

Mutations in the nonpolar side chains near histidine (His) 92 indicate that they form the residual structure of the alpha subunit of tryptophan synthase from Escherichia coli. A hydrophobic effect involving the side chains stabilizes the residual structure. The residual structure is seen in the presence of urea, when the secondary structure is disrupted. The C-epsilon proton of His 92 is sensitive to transitions between the state with the residual structure and the unfolded state. The transition process has negative enthalpy and entropy values at 25 degrees centigrade.

The three-dimensional structure of hemoglobin, in which the beta-globin Valine(super 67) is replaced with threonine, indicates small changes in conformation associated with the mutation. However, the mutation decreases the affinity of the mutant for oxygen, carbon monoxide, and nitric oxide. There are no water molecules associated with the threonines in the beta-subunits. There is an increase in polarity of the heme pocket, which affects its function and stability.

The interactions of the chicken egg white lysozyme's asparagine 46 (Asn46) and aspartate 52 (Asp52) with the substrate are responsible for the stability of intermediate states and complexes formed with cell wall substrates. Mutations in either Asn46 or Asp52 to Ala affect the interactions of the other residue with the substrate suggesting the presence of a hydrogen bond between the residues. The mutations have no effect on the dissociation constants of complexes with chitin. The complexes with chitin appear to differ in conformation from the complexes with bacterial peptidoglycans.

The catalytic mechanism of glucoamylase appears to involve four steps, and the catalytic base Glu400 is necessary to strengthen binding at subsite 1. Mutations in the residues near the enzyme's active site reduce the enzymatic activity for alpha-D-glucosyl fluoride (GF) and maltose. The replacement of the active site's Tyr116 and Trp120 residues reduces activity only for maltose. The interaction with the substrate aglycon part is probably a rate-limiting step. The binding of GF to a Glu400 mutant appears to deprotonate a carboxyl group that enhances catalysis.

Overexpression of wild-type cystic fibrosis transmembrane conductance regulator (CFTR), a chloride (Cl-) channel, arrests cell growth in the G(sub 2)/M phase and increases the cell volume of monkey kidney cells. CFTR overexpression in the G551D-CFTR mutant inhibits growth slightly, and there is an increase in the number of cells in the G(sub 2)/M phase. Decrease in the threshold of Cl- channel activity in the mutant produces growth defects. CFTR is capable of downregulating its activity.

Liddle's disease is caused by abnormal activity of the peripheral blood lymphocytes' amiloride-sensitive Na+ channels due to a mutation in the beta-subunit of the channels. Na+ currents in individuals, affected with the genetic disease, are strongly activated and do not increase in the presence of 8-(-4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (cAMP). The cAMP-induced current is normalized by a peptide, consisting of deleted residues. In normal individuals the Na+ current is low. Amiloride inhibits the Na+ current in both affected and normal individuals.

Several G protein-coupled receptors promote the protein kinase A (PKA)-mediated phosphorylation of Na-K-adenosinetriphosphatase (Na-K-ATPase) in different cell systems. The phosphorylation inducing receptors are adrenergic, dopaminergic and muscarinic cholinergic receptor subspecies. Stimulation of the receptors induces phosphorylation of the wild-type and PKC phosphorylation site-deficient Na-K-ATPase. However, the Na-K-ATPase alpha subunit, lacking the PKA phosphorylation site, does not undergo phosphorylation.

The pink-eye and the white-eye gene loci are tightly linked in the mosquito, Anopheles gambiae. The loci exhibit a recombination frequency of 3.5% and 1.1%. Only two genes on the X chromosome influence eye color and are capable of undergoing mutations. Phenotypic similarity, chromosomal gene conservation, and the presence of molecular lesions in the white gene suggest that many sex-linked mutations in eye color are in white homologs. The white mutants, unlike the pink mutants, show epistasis on expression of the larval body pigmentation phenotype collarless(super +).

The BED1 gene of Saccharomyces cerevisiae is essential for polarized growth and efficient budding. BED1 encodes an alpha-1,2-galactosyltransferase homologue, an integral membrane protein present in the endoplasmic reticulum. The BED1 mutant cell neither exhibits bud emergence nor possesses the ellipsoidal shape specific for wild-types, and are bigger than the wild-type cells. An intact morphogenesis checkpoint control helps in the survival of the mutants. Polarized growth and bud emergence depend on the glycosylation of specific proteins or lipids.

The cysteine residue at position 289 is necessary for the catalytic activity of fatty aldehyde dehydrogenase (Vh.ALDH). Mutation in the residue inactivates the enzyme while the ability to bind nucleotides is retained. The Vh.ALDH gene encodes a 54.5 kDa polypeptide whose sequence is different from other aldehyde dehydrogenases. Absorption and fluorescence analyses of NAD(P)H reveal that NAD+ and NADP+ attach to the same site and saturation of Vh.ALDH with NADP+ occurs with a Michaelis constant 40 times less than the other aldehyde dehydrogenases. The specificity of Vh.ALDH towards NADP+ is high.

Continuity of domains is important for protein stability but is not essential for the proteins to attain a native-like functional structure. A polymerase chain reaction mechanism establishes two mutants of yeast phosphoglycerate kinase with variations in the N- and C-terminal domains. The mutants fold in a conformation with little variations in the secondary and tertiary structures. The circular permutations alter the structure and stability of proteins to some extent.

The replacement of an amino acid in the oxyanion hole of papain by a glutamic acid residue confers nitrile hydratase activity to the cysteine protease. The Gln19Glu mutant enzyme has a nonionized side chain and its activities are similar to that of the wild-type enzyme. The mutant hydrolyzes the substrate to an amide. The normal amidase activity of papain converts the amide into nitrile.

The residues at positions P(sub 10) and P(sub 12) in the hinge region of alpha(sub 1)-antitrypsin are important for the inhibitory action on elastase. The enzyme has small side-chain amino acids such as alanine in the P(sub 8) to P(sub 12) hinge region. Mutations in the amino acids reveal that the substitutions at positions P(sub 7), P(sub 8), P(sub 9) and P(sub 11) cause no effect on the inhibitory action. Substitution at P(sub 10) allows the formation of the elastase-serpin complex which breaks down to cleave the enzyme. Substitution at P(sub 12) reduces the inhibitory action.

A study analyzes the substitutions in the protease domain of apolipoprotein(a) (apo(a)) and the replacement of Arg with Ser inactivate apo(a). The study uses a mutant with a substitution in the Arg residue containing a Ser residue to observe the proteolytic activity of a recombinant form of apo(a). Cleavage of the mutant with tissue-type plasminogen activator fails to affect the activity. The presence of a number of kringle IV repeats seems to have no effect on apo(a) and the protease region remains unmodified.

A mutation in the Leu residue at position 28 with Phe enhances hydride transfer cooperatively in the dihydrofolate reductase from Escherichia coli. Leu28 and Leu54 form part of the substrate binding site of the enzyme. Single mutations with aromatic side chain residues increase the affinity for the substrate H2F. However, mutations in both the residues increase crowding or rigidity at the active site and decrease the affinity for the substrate.

The pp70/85-kDa S6 kinases, collectively referred to as [pp70.sup.S6k], are thought to participate in transit through the [G.sub.1] phase of the cell cycle. [pp70.sup.S6k] regulates the phosphorylation of the 40S ribosomal protein S6 and the transcription factor [CREM.sub.tau] [pp70.sup.S6k] is regulated by serine/threonine phosphorylation, and although 1-phosphatidylinositol 3-kinase and phospholipase C have been implicated as upstream regulators, the mechanism of activation and identity of the upstream [pp70.sup.S6k] kinases remain unknown. To improve our understanding of how this mitogen-stimulated protein kinase is regulated by growth factors and the immunosuppressant rapamycin, we have initiated a structure/function analysis of [pp70.sup.S6k] Our results indicate that both the N and C termini participate in the complex regulation of [pp70.sup.S6k] activity.

An efficient method for mapping mutations is described in which hybrid genes, derived partly from mutant and partly from wild-type DNA, are obtained in vivo by homologous recombination of multiple fragments. The Recombinants are formed in a strain in which their phenotypes are immediately apparent. This method was developed to identify changes that disrupt protein-protein interactions demonstrable by the two-hybrid system in yeast. However, it can be extended to any system where recombination is possible, provided an assay is available to distinguish between mutant and wild-type phenotypes.

Although most nuclear hormone receptors are ligand-dependent transcriptional activators, certain members of this superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR), are involved in transcriptional repression. The silencing function of these receptors has been localized to the ligand binding domain (LBD). Previously, we demonstrated that overexpression of either the entire LBD or only the N-terminal region of the LBD (amino acids 168-259) is able to inhibit the silencing activity of TR. From this result we postulated the existence of a limiting factor (corepressor) that is necessary for TR silencing activity. To support this hypothesis, we identified amino acids in the N-terminal region of the LBD of TR that are important for the corepressor interaction and for the silencing function of TR. The silencing activity of TR was unaffected by overexpression of the LBD of mutant TR (VI74A/D177A), suggesting that valine at position 174 and/or aspartic acid at position 177 are important for corepressor interaction. This mutant receptor protein, V174/dl77, also lost the ability to silence target genes, suggesting that these amino acids are important for silencing function. Control experiments indicate that this mutant TR maintains its wild-type hormone binding and transactivation functions. These findings further strengthen the idea that the N-terminal region of the LBD of TR interacts with a putative corepressor protein(s) to achieve silencing of basal gene transcription.

The isocitrate dehydrogenase of Escherichia coli, which lacks the Rossmann fold common to other dehydrogenases, displays a 7000-fold preference for NADP over NAD (calculated as the ratio of [k.sub.cat]/[K.sub.m). Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to introduce six substitutions in the adenosine binding pocket that systematically shift coenzyme preference toward NAD. The engineered enzyme displays an 850-fold preference for NAD over NADP, which exceeds the 140-fold preference displayed by a homologous NAD-dependent enzyme. Of the six mutations introduced, only one is identical in all related NAD-dependent enzyme sequences - strict adherence to homology as a criterion for replacing these amino acids impairs function. Two additional mutations at remote sites improve performance further, resulting in a final mutant enzyme with kinetic characteristics and coenzyme preference comparable to naturally occurring homologous NAD-dependent enzymes.

At alkaline pH the bacteriorhodopsin mutant D85N, with aspartic acid-85 replaced by asparagine, is in a yellow form ([[lambda].sub.max [double tilde] 405 nm) with a deprotonated Schiff base. This state resembles the M intermediate of the wild-type photocycle. We used time-resolved methods to show that this yellow form of D85N, which has an initially unprotonated Schiff base and which lacks the proton acceptor Asp-85, transports protons in the same direction as wild type when excited by 400-nm flashes. Photoexcitation leads in several milliseconds to the formation of blue (630 nm) and purple (580 nm) intermediates with a protonated Schiff base, which decay in tens of seconds to the initial state (400 nm). Experiments with pH indicator dyes show that at pH 7, 8, and 9, proton uptake occurs in about 5-10 ms and precedes the slow release (seconds). Photovoltage measurements reveal that the direction of proton movement is from the cytoplasmic to the extracellular side with major components on the millisecond and second time scales. The slowest electrical component could be observed in the presence of azide, which accelerates the return of the blue intermediate to the initial yellow state. Transport thus occurs in two steps. In the first step (milliseconds), the Schiff base is protonated by proton uptake from the cytoplasmic side, thereby forming the blue state. From the pH dependence of the amplitudes of the electrical and photocycle signals, we conclude that this reaction proceeds in a similar way as in wild type - i.e., via the internal proton donor Asp-96. In the second step (seconds) the Schiff base deprotonates, releasing the proton to the extracellular side.

Nitric oxide synthases (NOSs) require tetrahydrobiopterin ([BH.sub.4]) for dimerization and NO production. Mutation analysis of mouse inducible NOS (iNOS; NOS2) identified Gly-450 and Ala-453 as critical for NO production, dimer formation, and [BH.sub.4] binding. Substitutions at five neighboring positions were tolerated, and normal binding of heme, calmodulin, and NADPH militated against major distortions affecting the [NH.sub.2]-terminal portion, midzone, or COOH terminus of the inactive mutants. Direct involvement of residues 450 and 453 in the binding of [BH.sub.4] iS supported by the striking homology of residues 448-480 to a region extensively shared by the three [BH.ssub.4]-utilizing aromatic amino acid hydroxylases and is consistent with the conservation of these residues among all 10 reported NOS sequences, including mammalian NOSs 1, 2, and 3, as well as avian and insect NOSs. Altered binding of [BH.sub.4] and/or L-arginine may explain how the addition of a single methyl group to the side chain of residue 450 or the addition of three methylenes to residue 453 can each abolish an enzymatic activity that reflects the concerted function of 1143 other residues.

Liddle syndrome is a mendelian form of hypertension characterized by constitutively elevated renal Na reabsorption that can result from activating mutations in the [beta] or [gamma] subunit of the epithelial Na channel. All reported mutations have deleted the last 45-76 normal amino acids from the cytoplasmic C terminus of one of these channel subunits. While these findings implicate these terminal segments in the normal negative regulation of channel activity, they do not identify the amino acid residues that are critical targets for these mutations. Potential targets include the short highly conserved Pro-rich segments present in the C terminus of [beta] and [gamma] subunits; these segments are similar to SH3-binding domains that mediate protein-protein interaction. We now report a kindred with Liddle syndrome in which affected patients have a mutation in codon 616 of the [beta] subunit resulting in substitution of a Leu for one of these highly conserved Pro residues. The functional significance of this mutation is demonstrated both by the finding that this is a de novo mutation appearing concordantly with the appearance of Liddle syndrome in the kindred and also by the marked activation of amiloride-sensitive Na channel activity seen in Xenopus oocytes expressing channels containing this mutant subunit (8.8-fold increase compared with control oocytes expressing normal channel subunits; P = 0.003). These findings demonstrate a de novo missense mutation causing Liddle syndrome and identify a critical channel residue important for the normal regulation of Na reabsorption in humans.

Isolated Clostridium butyricum DSM 5431 mutants have increased resistance to glycerol and 1,3-propanediol (1,3-PD) and produce more biomass compared to the wild type. The wild-type strain produces more acid than the mutant strain and releases more acetic acid than butyrate. In contrast to the wild type, the mutants use more NADH to form 1,3-PD. The mutant strains have low levels of hydrogenase and NADH-ferrodoxin activities and an increased level of ferrodoxin-NAD+ reductase activity.

Arabidopsis revertant lines formed from mutant plants show epigenetic gene silencing by hypomethylation of the phosphoribosylanthranilate isomerase (PAI) genes. The mutants contain a deletion mutation so that the copy number of the PAI genes is reduced to two, giving rise to a fluorescent phenotype. The mutation is meiotically heritable but is unstable and wild-type revertant lines are formed. The hypomethylation is active over the entire genome and is not restricted to specific chromosomal regions.

A differentiating system based on embryonic stem (ES) cell-derived embryoid bodies (EBs) which recapitulates the in vivo cardiac, skeletal, and smooth muscle myogenesis of mouse embryos was developed and used to investigate the effects of the disruption of the desmin gene on muscle cell differentiation. Wild-type, heterozygous, and homozygous cell lines with the mutated desmin allele were evaluated. Skeletal myogenesis was totally inhibited in desmin null mutant Ebs, as manifested by the absence of myotube formation, contractility, and myoD, myogenin, myf5, and myosin heavy chain expression. Smooth muscle formation was also completely blocked in the absence of desmin. On the other hand, there were no obvious effects on cardiomyocyte differentiation in these desmin null mutant EBs. However, reduced desmin expression in EBs heterozygous for the desmin mutation leads to partial inhibition of cardiac muscle formation. These data suggest that in contrast to early cardiocyte differentiation, desmin is indispensable for skeletal and smooth muscle formation.

Asp 46 can replace Asp 96 as a Schiff base proton donor in the photocycle of bacteriorhodopsin (bR). A series of metabolic intermediates with different visible absorption are formed on absorption of a proton by bR, of which the M to N transition is studied. A study of the double mutation Thr 46 to Asp/Asp 96 to Asn (T46D/D96N), the single mutants Asp 96 to Asn (D96N) and Thr 46 to Asp indicate that T46D/D96N has M decay values that are similar to the wild-type. Bands accompanying structural changes of Asn 96 in the mutant D96N are lacking in T46D/D96N.

The imidazole group of His at position 52 in the yeast enzyme, cytochrome c peroxidase, CcPCN, is necessary for catalysis. NMR spectroscopic studies of a mutant in which Asn82 is replaced with Ala indicate that the mutation disrupts a hydrogen bond between His52 and the heme-ligated CN. The heme active site of the enzyme appears to be microheterogenous. The enzyme preparation has two magnetically inequal forms that interconvert with changes in temperature. The equilibrium constant of the enzyme is 1.5 at 20 degrees celsius.

Bacteriorhodopsin is a membrane protein that functions as a light-driven proton pump. Each cycle of proton transport is initiated by the light-induced isomerization of retinal from the all-trans to 13-cis configuration and is completed by the protein-driven reisomerization of retinal to the all-trans configuration. Previous studies have shown that replacement of Leu-93, a residue in close proximity to the 13-methyl group of retinal, by alanine, resulted in a 250-fold increase in the time required to complete each photocycle. Here, we show that the kinetic defect in the photocycle of the Leu-93 [arrow right] Ala mutant occurs at a stage after the completion of proton transport and can be overcome in the presence of strong background illumination. Time-resolved retinal-extraction experiments demonstrate the continued presence of a 13-cis intermediate in the photocycle of the Leu-93 [arrow right] Ala mutant well after the completion of proton release and uptake. These results indicate that retinal reisomerization is kinetically the rate-limiting step in the photocycle of this mutant and that the slow thermal reisomerization can be bypassed by the absorption of a second photon. The effects observed for the Leu-93 [arrow right] Ala mutant are not observed upon replacement of any other residue in van der Waals contact with retinal or upon replacement of Leu-93 by valine. We conclude that the contact between Leu-93 and the 13-methyl group of retinal plays a key role in controlling the rate of protein conformational changes associated with retinal reisomerization and return of the protein to the initial state.

Ribosomal DNA (rDNA) located in place of the promoter of a variant surface glycoprotein (VSG) expression site of Trypanosoma brucei (T. brucei) has the same functions as the VSG promoters. The T. brucei variants containing rDNA are infective and similar to the wild-type parasites in human blood. However, the structure of the rDNA and the VSG promoter are different. The switching between different VSG expression sites is probably due to telomeric silencing. The suppression of the VSG expression sites occurs during transcription initiation.

The effects of the rotational information of DNA in determining the in vitro localization of nucleosomal core particles (ncps) have been studied in the Saccharomyces cerevisiae 5S rRNA repeat gene. We have altered the distribution of the phased series of flexibility signals present on this DNA by inserting a 25-bp tract, and we have analyzed the effects of this mutation on the distribution and on the frequencies of ncps, as compared with the wild type and a reference 21-bp insertion mutant. The variation of the standard free energy of nucleosome reconstitution was determined. The results show that the DNA rotational information is a major determinant of ncps positioning, define how many rotationally phased signals are required for the formation of a stable particle, and teach how to modify their distribution through the alteration of the rotational signals.

The MKC7 gene was isolated as a multicopy suppressor of the cold-sensitive growth phenotype of a yeast kex2 mutant, which lacks the protease that cleaves pro-[alpha]-factor and other secretory proproteins at pairs of basic residues in a late Golgi compartment in yeast. MKC7 encodes an aspartyl protease most closely related to product of the YAP3 gene, a previously isolated multicopy suppressor of the pro-[alpha]-factor processing defect of a kex2 null. Multicopy MKC7 suppressed the [alpha]-specific mating defect of a kex2 null as well as multicopy YAP3 did, but multicopy YAP3 was a relatively weak suppressor of kex2 cold sensitivity. Overexpression of MKC7 resulted in production of a membrane-associated proteolytic activity that cleaved an internally quenched fluorogenic peptide substrate on the carboxyl side of a Lys-Arg site. Treatment with phosphatidylinositol-specific phospholipase C shifted Mkc7 activity from the detergent to the aqueous phase in a Triton X-114 phase separation, indicating that membrane attachment of Mkc7 is mediated by a glycosylphosphatidylinositol anchor. Although disruption of MKC7 or YAP3 alone resulted in no observable phenotype, mkc7 yap3 double disruptants exhibited impaired growth at 37[degrees]C. Disruption of MKC7 and YAP3 in a kex2 null mutant resulted in profound temperature sensitivity and more generalized cold sensitivity. The synergism of mkc7, yap3, and kex2 null mutations argues that Mkc7 and Yap3 are authentic processing enzymes whose functions overlap those of Kex2 in vivo.

The plant acyl-acyl carrier protein (ACP) thioesterases (TEs) are of biochemical interest because of their roles in fatty acid synthesis and their utilities in the bioengineering of plant seed oils. When the FatB1 cDNA encoding a 12:0-ACP TE (Uc FatB1) from California bay, Umbellularia californica (Uc) was expressed in Escherichia coli and in developing oilseeds of the plants Arabidopsis thaliana and Brassica napus, large amounts of laurate (12:0) and small amounts of myristate (14:0) were accumulated. We have isolated a TE cDNA from camphor (Cinnamomum camphorum) (Cc) seeds that shares 92% amino acid identity with Uc FatB1. This TE, Cc FatB1, mainly hydrolyzes 14:0-ACP as shown by E. coli expression. We have investigated the roles of the N- and C-terminal regions in determining substrate specificity by constructing two thimeric enzymes, in which the N-terminal portion of one protein is fused to the C-terminal portion of the other. Our results show that the C-terminal two-thirds of the protein is critical for the specificity. By site-directed mutagenesis, we have replaced several amino acids in Uc FatB1 by using the Ce FatB1 sequence as a guide. A double mutant, which changes Met-197 to an Arg and Arg.;199 to a His (MI97R/RL99H), turns Uc FatB1 into a 12:0/14:0 TE with equal preference for both substrates. Another mutation, T231K, by itself does not effect the specificity. However, when it is combined with the double mutant to generate a triple mutant (MI97R/RL99H/T231K), Uc FatB1 is converted to a 14:0-ACP TE. Expression of the double-mutant EDNA in E. coli K27, a strain deficient in fatty acid degradation, results in accumulation of similar amounts of 12:0 and 14:0. Meanwhile the E. coli expressing the triplemutant cDNA produces predominantly 14:0 with very small amounts of 12:0. Kinetic studies indicate that both wild-type Uc FatB1 and the triple mutant have similar values of [K.sub.m,app] with respect to 14:0-ACP. Inhibitory studies also show that 12:0-ACP is a good competitive inhibitor with respect to 14:0-ACP in both the wild type and the triple mutant. These results imply that both 12:0- and 14:0-ACP can bind to the two proteins equally well, but in the case of the triple mutant, the hydrolysis of 12:0-ACP is severely impaired. The ability to modify TE specificity should allow the production of additional 'designer oils' in genetically engineered plants.