Silent Mutants Research Articles

Electrically silent mutants unravel the mechanism of binding-gating coupling in Cys-loop receptors.

The transduction of extracellular chemical signals into intracellular events relies on the communication between neighboring domains of membrane receptors. In the particular case of Cys-loop receptor channels, five short stretches of amino acids, one per subunit, link the extracellular and transmembrane domains in such a way that the ion permeability of the latter and the affinity for neurotransmitters of the former become tied to each other. Here, using direct functional approaches, we set out to understand the molecular bases of this crucial interdependence through the characterization of total loss-of-current mutations at the interface between domains. Our results indicate that domain-domain proximity plays a previously unnoticed critical role inasmuch as inserting a single residue in each linker rendered the two domains independent of each other. In marked contrast, loss-of-current mutations that leave the linkers' length unaltered did not compromise the interdomain coupling, but rather, seemed to cause agonist-bound closed receptors to desensitize without appreciably opening.

Acetylation of ANXA1 reduces caspase-3 activation by enhancing the phosphorylation of caspase-9 under OGD/R conditions

SIRT2, a Class III HDACs, aggravates cell damage and activates caspase-3 under oxygen-glucose deprivation/reoxygenation and glucose (OGD/R) conditions. In this paper, we demonstrated the adverse effects of SIRT2 on cells after OGD/R attacks, which were mediated by increased interactions between SIRT2 and ANXA1, and explicated the mechanisms by which acetylated ANXA1 affects the activation and cleavage of caspase-3. We found that the acetylation level of ANXA1 was decreased through the its increased interactions with SIRT2 after the OGD/R insult. The lysine 312 residue (K312) was selected as the target site in ANXA1 because it is associated with SIRT2, and its mimic (K312Q) and silent (K312R) mutants were then established through site mutagenesis. Under OGD/R conditions, the acetylation mimic of K312Q ANXA1 accumulated in the cytoplasm, decreasing the activity levels of caspase-3 and the upstream initiator caspase-9, compared with the levels of WT and K312R ANXA1. Furthermore, K312Q ANXA1 intervened in the interactions of caspase-3 to caspase-9 by increasing the phosphorylation levels of caspase-9 and inhibited its cleavage by downregulating PRKAR2B, a regulatory subunit of protein kinase A (PKA). In this process, K312Q ANXA1 was found to be directly associated with PRKAR2B, diminishing its restriction on the catalytic subunit of PKA.In conclusion, acetylated ANXA1 can promote the phosphorylation of caspase-9 to decrease the activation of caspase-3 by enhancing the expression of a kinase upstream of caspase-9 after the OGD/R stimulation.

Validation of Omega Subunit of RNA Polymerase as a Functional Entity.

The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ’ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, β’, and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, E. coli mutants lacking rpoZ (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ββ’ subassembly. We observed that the interaction between α2ββ’ and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in rpoZ. We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code’s position in the reading frame of rpoZ plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ββ’. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the β’ subunit, and it is the same for both types of mutants.

Unraveling the Role of Silent Mutation in the ω-Subunit of Escherichia coli RNA Polymerase: Structure Transition Inhibits Transcription.

The bacterial RNA polymerase is a multi-subunit enzyme complex composed of six subunits, α2ββ’σω. The function of this enzyme is to transcribe the DNA base sequence to the RNA intermediate, which is ultimately translated to protein. Though the contribution of each subunit in RNA synthesis has been clearly elucidated, the role of the smallest ω-subunit is still unclear despite several studies. Recently, a study on a dominant negative mutant of rpoZ has been reported in which the mutant was shown to render the RNA polymerase defective in transcription initiation (ω6, N60D) and gave an insight on the function of ω in RNA polymerase. Serendipitously, we also obtained a silent mutant, and the mutant was found to be lethal during the isolation of toxic mutants. The primary focus of this study is to understand the mechanistic details of this lethality. Isolated ω shows a predominantly unstructured circular dichroism profile and becomes α-helical in the enzyme complex. This structural transition is perhaps the reason for this lack of function. Subsequently, we generated several silent mutants of ω to investigate the role of codon bias and the effect of rare codons with respect to their position in rpoZ. Not all silent mutations affect the structure. RNA polymerase when reconstituted with structurally altered silent mutants of ω is transcriptionally inactive. The CodonPlus strain, which has surplus tRNA, was used to assess for the rescue of the phenotype in lethal silent mutants.

Expression, Purification, and Activity of ActhiS, a Thiazole Biosynthesis Enzyme from Acremonium chrysogenum.

Thiamine pyrophosphate is an essential coenzyme in all organisms. Its biosynthesis involves independent syntheses of the precursors, pyrimidine and thiazole, which are then coupled. In our previous study with overexpressed and silent mutants of ActhiS (thiazole biosynthesis enzyme from Acremonium chrysogenum), we found that the enzyme level correlated with intracellular thiamine content in A. chrysogenum. However, the exact structure and function of ActhiS remain unclear. In this study, the enzyme-bound ligand was characterized as the ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2-carboxylic acid (ADT) using HPLC and 1H NMR. The ligand-free ActhiS expressed in M9 minimal medium catalyzed conversion of NAD+ and glycine to ADT in the presence of iron. Furthermore, the C217 residue was identified as the sulfur donor for the thiazole moiety. These observations confirm that ActhiS is a thiazole biosynthesis enzyme in A. chrysogenum, and it serves as a sulfur source for the thiazole moiety.

α Globin Hemoglobinopathy as A Case Study: Mutants and Prototype Mutants: Are They Absent α0, Reduced α+ or Recombinant?

Recently the existence of prototype mutants in β globin genes was proved. In this study a further and similar investigation was performed. The investigation concerns the Homo sapiens α globin. The prototype mutants were screened in the α globin DNA sequences. The results proved their existence. A selected α globin fragment contains the prototype mutant was used for searching both of the nucleotide and protein databases. The used fragment consists of nine nucleotides GGC AAG GTT and contain two silent mutants with the following amino acids sequences GKV. Searching the Blast protein database for the existence of correct mutants using the V00493.7 Homo sapiens 27 amino acids fragment was led to find 50 different sequences. Interestingly the protein sequences show similarity with marsupials, even-toed ungulates, odd-toad ungulates, placental, bats, rodents, carnivores, whales and dolphins; primates, insectivores, rabbits and hares. Species responsible for human bit or blood transfer are located and suggest gene recombination between different species occure through blood transfer. The restriction map for the 81 nucleotides for four nucleotide sequences (one prototype mutant, two correct mutants, and one normal) proved change in the restriction enzymes profile of the α globin sequences. That might be a proof for the existence of reduced α+ globin (also in case of β+ globin) macromolecules. The protein modelling in the selected different fragments show differences in the 3D structure and in their hydrophobicity. The unexpected exo-sources for the α globin put another load on the possibility of the haemoglobin illness solving. However, avoiding marriage from relatives, and from the same ethnic groups might be the best choice. Additionally avoiding blood contact with animals have compatible mutants is recommended. Further investigations are in need to prove/disprove the role of the gene recombination between species through blood in the transfer of the α globin mutants or even other ones.

Genetic Code-guided Protein Synthesis and Folding in Escherichia coli

Universal genetic codes are degenerated with 61 codons specifying 20 amino acids, thus creating synonymous codons for a single amino acid. Synonymous codons have been shown to affect protein properties in a given organism. To address this issue and explore how Escherichia coli selects its "codon-preferred" DNA template(s) for synthesis of proteins with required properties, we have designed synonymous codon libraries based on an antibody (scFv) sequence and carried out bacterial expression and screening for variants with altered properties. As a result, 342 codon variants have been identified, differing significantly in protein solubility and functionality while retaining the identical original amino acid sequence. The soluble expression level varied from completely insoluble aggregates to a soluble yield of ~2.5 mg/liter, whereas the antigen-binding activity changed from no binding at all to a binding affinity of > 10(-8) m. Not only does our work demonstrate the involvement of genetic codes in regulating protein synthesis and folding but it also provides a novel screening strategy for producing improved proteins without the need to substitute amino acids.

Awakening Loss-of-Function KATP Channel Mutants with an Engineered ‘Forced Gating’ Approach

Regulation of inwardly-rectifying potassium channels by intracellular ligands couples membrane excitability to important signaling cascades and metabolic pathways. A significant barrier to understand coupling mechanisms between ligand binding and gating is that many functionally important channel motifs are highly sensitive to mutagenesis, resulting in a loss of function phenotype. We have developed a ‘forced gating’ approach that rescues function in electrically silent channel mutants, enabling characterization of channel motifs that are otherwise intractable to electrophysiological recording. This approach involves substitution of a glutamate in the hydrophobic Kir channel bundle crossing (F168E mutation in Kir6.2), generating channels that are pH sensitive and open upon alkalization, due to mutual repulsion of introduced negatively charged side chains in the channel gate. We have implemented this ‘forced gating’ approach in mutagenic scans of the Kir channel slide helix and G-loop, two motifs proposed to play a role in ligand dependent gating of Kir channels. Both motifs are also highly sensitive to mutagenesis, with alanine mutations causing nearly complete loss-of-function at 7/20 slide helix positions, and 8/13 G-loop positions. Without exception, expression of silent mutants on the Kir6.2[F168E] background permitted activation of functional channels in alkaline pH, and measurement of kinetics and potency of ATP inhibition. Our results highlight an essential ‘aspartate anchor’ (Kir6.2 residue D58) that bridges the slide helix and multiple interacting residues in the cytoplasmic domain. Disruption of the highly conserved ‘aspartate anchor’ uncouples the transmembrane and cytoplasmic domains, reducing ATP sensitivity of Kir6.2 far more than any other G-loop or slide helix mutants. These findings indicate a central role for the ‘aspartate anchor’ in coupling ligand binding to Kir gating, and also emphasize the potential general utility of this ‘forced gating’ method to study loss-of-function channel mutants.

Glucose Catabolism with Resultant Formation of Reactive Oxygen Species and Local Hypoxia Modulates the Induction and Expansion of Hematopoietic Stem Cells

AbstractAbstract 408Metabolic disorders, including obesity, diabetes, and their related complications have become the leading cause of preventable death in the U.S. While effects of increased blood sugar on the cardiovascular system and other organs are well established, consequences for the hematopoietic system are less well characterized. Specifically, the effects of gestational diabetes on embryonic blood formation have not been elucidated in detail. To determine the impact of elevated glucose concentrations on hematopoietic stem cell (HSC) induction and expansion, we treated zebrafish embryos with D-glucose from 12 to 36 hours post fertilization (hpf). HSCs in the Aorta-Gonad-Mesonephros (AGM) region were expanded in a dose-responsive manner, as assessed by in situ hybridization for the conserved HSC markers, runx1 and cmyb; this effect was verified by analysis of CD41 expression (n≥25–50 embryos/condition). A significant 3-fold enhancement in HSC number was observed after 1% glucose treatment by qPCR for runx1 and FACS analysis of fluorescent HSC reporter embryos, Tg(cmyb:eGFP) and Tg(-6.0itga2b:eGFP)la2 (CD41:GFP). Other mono-, di-, and tri-saccharides exerted similar effects, expanding the number of HSCs. However, L-glucose, the inactive enantiomer, did not affect runx1 expression, implying that the effect of the metabolizable saccharides is independent of osmotic dynamics. Furthermore, treatment with glucose from 12 to 120 hpf resulted in a sustained increase in HSC number throughout the duration of treatment. Increased cellular proliferation was observed in the AGM region following 1% glucose treatment, as assessed by elevated BrdU incorporation and cyclin D1 expression. Additionally, acridine orange staining revealed a decrease in apoptotic cell number. HSC formation was impaired by morpholino knockdown of the glucose transporter glut1, indicating the requirement of glucose uptake for HSC formation. Exposure to chemical inhibitors of glycolysis (ethyl-3-bromopyruvate) and of oxidative phosphorylation (cyanide and oxaloacetate) reversed the beneficial effects of glucose on HSCs. In contrast, pharmacological and genetic modulations of the metabolic endocrine hormones IGF and insulin did not alter the effects of glucose treatment. Treatment with the glycolytic intermediate pyruvate expanded HSCs, while exposure to glucosamine, the first component of the hexosamine biosynthetic pathway, had no effect on blood stem cells. These results suggest that glucose catabolism specifically is responsible for HSC expansion. A potential consequence of heightened glucose metabolism is increased generation of reactive oxygen species (ROS), which may serve as important signaling factors mediating the observed effects on HSCs. In support of this hypothesis, treatment with the antioxidant N-acetylcysteine, which alone decreased HSC formation, could not be rescued by concomitant glucose exposure, while the oxidant, 1-phenyl-2-thiourea, elicited an expansion in HSC number. Using the fluorescent ROS sensor, dihydroethidium, we observed ROS in circulating erythrocytes, a subpopulation of CD41+ cells and in the adjacent somitic muscle. ROS are known inducers of the hypoxia sensor, hif1α, which regulates important hematopoietic genes including vegf and epo, as well as the glut1 glucose transporter, indicating a potential feedback loop controlling HSC induction. Chemical induction of hypoxia by cobalt chloride similarly enhanced HSC formation. Intriguingly, excess glucose can overcome the reduction of HSC number normally observed in silent heart mutants lacking blood circulation, possibly by ROS/hif1α-mediated induction of vegf and NO in the hematopoietic niche. The beneficial effect of glucose on hematopoiesis was conserved in adult zebrafish: FACS analysis of kidney marrow following sublethal irradiation revealed a more rapid recovery of the progenitor population after glucose exposure. Our data suggest that, in conjunction with the cardiovascular system, energy metabolism plays a key role in regulating hematopoetic induction and homeostasis. These results could lead to novel therapeutic approaches for HSC modulation, and may unveil specific risks of obesity and diabetes for hematopoiesis during gestation and in the adult. (equal contribution: WG, TEN). Disclosures:Goessling:Fate Therapeutics: Consultancy, Patents & Royalties. North:Fate Therapeutics: Consultancy, Patents & Royalties.

Role of Survivin Phosphorylation by Aurora B in Mitosis

The chromosomal protein passenger complex, a key mitotic regulator, consists of at least four proteins, INCENP, Aurora B, Survivin and Borealin. Survivin, in contrast to the other members of the chromosomal protein passenger complex (CPC), is mobile at metaphase. This protein is also phosphorylated by Aurora B at Threonine 117. In this work we have studied the role of the phosphorylation of Survivin in mitotosis by using non phosphorylable T117A and phosphomimic T117E silent resistant Survivin mutants, inducible cell lines expressing these mutants and a combination of siRNA, time-lapse microscopy and FRAP analysis. Time lapse microscopy and FRAP analysis show that Survivin T117A mutant is very stably associated with centromeres and its expression induces a prometaphasic arrest in endogenous survivin depleted cells. In addition, Survivin T117A was unable to rescue the phenotypes of the endogenous survivin depleted cells. Expressed in these cells, the phosphomimic Survivin T117E mutant exhibits a very weak interaction with the centromeres and behaves as a dominant negative mutant inducing severe mitotic defects. Our data suggest that the Aurora B generated phosphorylation/dephosphorylation cycle of Survivin is required for proper proceeding of mitosis.

Simulated Point Mutations in the Aα-Chain of Human Fibrinogen Support a Role of the αC Domain in the Stabilization of Fibrin Gel

The hydrophobicity pattern distribution in the Aα-, Bβ- and γ-chains of human fibrinogen has been studied by a nonlinear method, recurrence quantification analysis, in the wild type and in a number of naturally occurring or simulated mutants. The aim was to find a structural basis for distinguishing between silent and pathological mutants. We were successful in the case of mutations on the Aα-chain, thanks to the peculiar features of this chain as compared to the other two. Relevant findings concerning the point mutants of the Aα-chain are the following: (a) the recurrence quantification analysis-based classification of such mutants is in good agreement with the clinical classification, and (b) the location of the mutated residue on the sequence plays a more relevant role than its hydrophobic features. Artificial point mutants in the terminal zone (600–866 residues) of the extended isoform of the Aα-chain cluster together with the natural hemorrhagic mutants of the first (1–207) residues.

Application of metabolome data in functional genomics: A conceptual strategy

A gene with yet unknown physiological function can be studied by changing its expression level followed by analysis of the resulting phenotype. This type of functional genomics study can be complicated by the occurrence of ‘silent mutations’, the phenotypes of which are not easily observable in terms of metabolic fluxes (e.g., the growth rate). Nevertheless, genetic alteration may give rise to significant yet complicated changes in the metabolome. We propose here a conceptual functional genomics strategy based on microbial metabolome data, which identifies changes in in vivo enzyme activities in the mutants. These predicted changes are used to formulate hypotheses to infer unknown gene functions. The required metabolome data can be obtained solely from high-throughput mass spectrometry analysis, which provides the following in vivo information: (1) the metabolite concentrations in the reference and the mutant strain; (2) the metabolic fluxes in both strains and (3) the enzyme kinetic parameters of the reference strain. We demonstrate in silico that changes in enzyme activities can be accurately predicted by this approach, even in ‘silent mutants’.

Identification of the Geometric Requirements for Allosteric Communication between the α- and β-Subunits of Tryptophan Synthase

The pyridoxal 5'-phosphate-dependent tryptophan synthase alpha2beta2 complex is a paradigmatic protein for substrate channeling and allosteric regulation. The enzymatic activity is modulated by a ligand-mediated equilibrium between open (inactive) and closed (active) conformations of the alpha- and beta-subunit, predominantly involving the mobile alpha loop 6 and the beta-COMM domain that contains beta helix 6. The alpha ligand-triggered intersubunit communication seems to rely on a single hydrogen bond formed between the carbonyl oxygen of betaSer-178 of beta helix 6 and the NH group of alphaGly-181 of alpha loop 6. We investigated whether and to what extent mutations of alphaGly-181 and betaSer-178 affect allosteric regulation by the replacement of betaSer-178 with Pro or Ala and of alphaGly-181 with either Pro to remove the amidic proton that forms the hydrogen bond or Ala, Val, and Phe to analyze the dependence on steric hindrance of the open-closed conformational transition. The alpha and beta activity assays and the equilibrium distribution of beta-subunit catalytic intermediates indicate that mutations do not significantly influence the intersubunit catalytic activation but completely abolish ligand-induced alpha-to beta-subunit signaling, demonstrating distinct pathways for alpha-beta-site communication. Limited proteolysis experiments indicate that the removal of the interaction between betaSer-178 and alphaGly-181 strongly favors the more trypsin-accessible open conformation of the alpha-active site. When the hydrogen bond cannot be formed, the alpha-subunit is unable to attain the closed conformation, and consequently, the allosteric signal is aborted at the subunit interface.

Non-redundant Role of Shc in Erk Activation by Cytoskeletal Reorganization

We have shown previously that cytoskeletal reorganization (CSR) induced by pharmacological reagents such as colchicine or cytochalasins can up-regulate the urokinase-type plasminogen activator (uPA) gene via the Ras/Erk signaling pathway. In this present study using the small interfering RNA technique, we have found that ShcA adapter proteins play a rather active role in CSR-induced Erk activation, contrary to their mostly redundant role in other signaling pathways, e.g. growth factor-induced Erk activation, where Grb2 can bind directly to the receptor tyrosine kinase and activate Erk in the absence of ShcA. ShcA knockdown abolished CSR-induced activation of both Erk and the uPA promoter. Expression of small interfering RNA-escaping silent mutants of p52 or p46 but not p66 ShcA isoform efficiently rescued CSR-induced Erk activation. Moreover, we have shown that phosphorylation of either Tyr-239/Tyr-240 or Tyr-313 in p52(ShcA) can mediate CSR-induced Erk activation equally well. In a quest for molecules upstream of ShcA in this signaling, we found that CSR-induced ShcA tyrosine phosphorylation, its association with Grb2, Erk activation, and uPA gene expression were all dependent on Rho kinase, p38 mitogen-activated protein kinase, and Src. In summary, we have found a novel, non-redundant role for ShcA in contrast to its redundant role in many other signaling pathways.

Role of Nucleotide Sequences in the V3 Region in Efficient Replication of CCR5-Utilizing Human Immunodeficiency Virus Type 1 in Macrophages

Macrophages express both CXCR4 and CCR5 coreceptors, but restrict X4 HIV-1 replication unless the Env-V3 region, a major determinant of cell tropism, is exchanged with that of R5 HIV-1. As the V3 exchange concomitantly alters the nucleotide sequences, we introduced silent mutations in the V3 or C2 region of macrophage-tropic R5 JRFL without changing the amino acids. Immunoblot analysis confirmed that viral proteins including Env-gp120 were similarly incorporated in wild-type (wt) and mutant virions. The silent mutants infected CCR5-positive MAGIC5 cells but not CCR5-negative MAGI cells, as productively as wt viruses, indicating that the silent mutations did not alter coreceptor utilization. In contrast, two of three silent V3-mutant viruses failed to replicate efficiently in primary macrophages, whereas other V3- or C2-mutants and wt JRFL infected macrophages productively. Furthermore, synthesis of the full-length viral DNA of the aberant V3-mutant was largely reduced in macrophages. These results suggest that V3 nucleotide sequences may be one of the postentry factors restricting HIV-1 replication in macrophages.

Electrically neutral microheterogeneity of human plasma transthyretin (prealbumin) detected by isoelectric focusing in urea gradients.

Mutants of the human plasma transthyretin (TTR, prealbumin) have attracted interest due to their rather frequent association with the autosomal dominant disease familial amyloidotic polyneuropathy (FAP). Some three quarters of known TTR mutations produce electrically neutral amino acid substitutions undetectable via separation by charge. We have developed an electrophoretic procedure sensitive to differences in the stability of tetramers and monomers under partially denaturing conditions. The differential folding states were found to be fully reversible. Applying the procedure we found 14 electrically silent mutants of TTR among 2000 plasma samples from German donors. We demonstrate that the normal TTR monomer exists in different forms of variable stability and/or charge due to binding of sulfhydryls from plasma to the unique cysteine at position 10 of the primary structure as well as due to modification by treatment with an oxidant. We found that reduction of Cys10 increases the stability of the folded monomeric and tetrameric conformations. The conformational changes of TTR induced by isoelectric focusing in a urea gradient were found to be associated by a gain of three positive charge units. Using published crystallographic data we present structural sites in the TTR molecule which could explain the observed effects.

Silent mutations in the Escherichia coli ompA leader peptide region strongly affect transcription and translation in vivo.

In order to test the effect of silent mutations on the regulation of gene expression, we monitored several steps of transcription and translation of the ompA gene in vivo , in which some or all codons between codons 6 and 14, frequently used in Escherichia coli , had been exchanged for infrequent synonymous codons. Northern blot analysis revealed an up to 4-fold reduction in the half-life of the mutated messengers and a >10-fold reduction in their steady-state amounts. Western blot analysis showed a 10-fold reduction in the amount of OmpA protein. Use of a system expressing a Rho-specific anti-terminator allowed us to detect a strong transcription polarity effect in the silent mutants. These results demonstrate that silent mutations can severely inhibit several steps of gene expression in E. coli and that code degeneracy is efficiently exploited in this species for setting signals for gene control and regulation.

Electrophoretically silent hemoglobin mutants as revealed by isoelectric focusing in immobilized pH gradients

The applications of isoelectric focusing in immobilized pH gradients to the analysis of (i) human hemoglobin mutants, (ii) animal hemoglobin mutants (from cattle, sheep, dog and mouse), and (iii) tryptic digests of alpha and beta chains, are discussed and evaluated. Immobilized pH gradients appear to be an excellent tool for screening of genetic polymorphism and for detecting "silent mutants", i.e. those substitutions involving amino acids with nonionizable side chains. At present, not even capillary zone electrophoresis, claimed to have a resolving power equivalent to 1 million theoretical plates, has shown a resolution capability comparable to that of immobilized pH gradients, at least in the field of protein separation.

The consequences of base-pair substitution mutations in AT- and GC-rich bacteria

The likely consequences, in terms of premature stop codons, detectable missense mutants, silent missense mutants, and degenerate codon changes, have been determined for all 12 individual base substitution changes. This has been done for the full, 61 sense codon, genetic code and also for the much more limited codon availabilities of AT- or GC-rich DNA. The specificities and outcomes of individual base substitutions are likely to be rather different at AT- or GC-rich extremes, and also from the situation at an intermediate DNA base-ratio where all 61 sense codons are available. In particular, at DNA base-ratio extremes many mutations will be to non-utilized codons, which may well act as nonsense mutants. These in turn will give novel classes of suppressor-containing revertants. Even in bacteria with intermediate DNA base-ratios, particular codons for a given amino acid may be favoured, over alternatives, because their use maximizes, or minimizes, the mutational consequences of one, or more, base substitution changes.

Application of anaerobic ion-exchange chromatography to the separation of hemoglobins in R from T conformational states.

As an allosteric protein, hemoglobin can assume at least two different quatenary structures, a deoxy or T for tense conformation and an oxy or R for relaxed conformation (2,3) depending upon its state of ligation. Because the pK values of oxy and deoxy- hemoglobins are different, molecules in the R conformation should behave differently than molecules in the T conformation on ion exchange chromatography. Kilmartin et al. (4) designed an anaerobic cation exchange chromatographic procedure with which they separated the deoxy hemoglobin from oxy hemoglobin and other R conformation hemoglobin components like CO-hemoglobin and sulfohemoglobin. In this report we describe the further development of Kilmartin's procedure to separate two electrophoretically silent hemoglobin mutants, Hb Potomac [beta 101 Glu leads to Asp] (5) a high oxygen affinity variant, and Hb M-Milwaukee [beta 67 Val leads to Glu] (6,8) a low oxygen affinity mutant from Hb A. The modifications we have made include (a) applying the hemolysate at a partial pressure of oxygen determined from an examination of the oxygen equilibrium curve to produce a favorable ratio of T to R conformations of the two hemoglobins to be separated, (b) controlling the partial pressure of oxygen tension of the column effluent during the development of the chromatogram.