An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes

Section: Methodsmentioning

confidence: 99%

Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer

Huang

Liu

Chen

et al. 2010

“…Construction of SERT Chimeras and Mutants-A PCR-based approach was chosen to generate rationally designed cross-species chimeras between hSERT and gSERT based on the method by Kirsch and Joly (37). The coding region of hSERT was previously cloned from human placenta and inserted into the pcDNA3 vector, denoted hSERT (33).…”

Section: Methodsmentioning

confidence: 99%

The Chicken Serotonin Transporter Discriminates between Serotonin-selective Reuptake Inhibitors

Larsen

Elfving

Wiborg

2004

The serotonin transporter (SERT) belongs to a family of sodium chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from extracellular spaces. SERT represents the main pharmacological target in the treatment of several clinical conditions, including depression and anxiety. Serotonin-selective reuptake inhibitors and tricyclic antidepressants are the most predominantly prescribed drugs in the treatment of depression. In addition to antidepressants also psychostimulants, like cocaine and amphetamines, are important SERT antagonists. In the present study, we report the cloning and characterization of chicken SERT. Although the uptake kinetic was very similar to human SERT, the pharmacological profiles differed considerably for the two species. We find that chicken SERT is capable of discriminating between different serotonin-selective reuptake inhibitors; thus, the potency of S-citalopram and paroxetine is reduced more than 40-fold. A cross-species chimera strategy was undertaken and followed by species-scanning mutagenesis. Differences in pharmacological profiles were tracked to amino acid residues 169, 172, and 586 in human SERT. Structure-activity studies on structurally related compounds indicated that species divergences in drug sensitivity between human and chicken SERT were arising from differences in coordination or recognition of an important aminomethyl pharmacophoric substructure, which is shared by all high affinity antidepressants. Consequently, we suggest that Ala 169 and Ile 172 of human SERT are important residues in sensing the N-methylation state of SERT antagonists.Serotonergic neurotransmission is modulated by active reuptake of serotonin (5-hydroxytryptamine or 5-HT) 1 from the extracellular space. Reuptake is mediated by the serotonin transporter (SERT), and 5-HT is subsequently either degraded by monoamine oxidases or repackaged into vesicles by the proton-dependent vesicular monoamine transporter. A balance between release and active reuptake of 5-HT determines the magnitude and duration of signaling and thus plays a key role in the spatio-temporal fine tuning of serotonergic neurotransmission.Malfunction in active 5-HT reuptake is speculated to be associated with psychiatric disorders, particularly anxiety and depression (1, 2). Antidepressants inhibiting 5-HT reuptake are used also in treatment of anxiety disorders, obsessivecompulsive disorders, and eating disorders (3, 4).SERT is a member of a large family of homologous integral membrane proteins (5). SERT is of considerable interest as a molecular target for many antidepressants, including the tricyclic antidepressants such as imipramine and amitriptyline, as well as the serotonin-selective reuptake inhibitors (SSRIs) like citalopram, fluoxetine, paroxetine, sertraline, and fluvoxamine. SERT is also target for drugs of abuse including cocaine and amphetamines such as 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy") (6, 7).SERT is a putative 12-transmembrane (TM) domain protein that co-transports...

“…Site-directed Mutagenesis of SsoII Variants-Site-directed mutagenesis of the SsoII gene was performed by a PCR-based technique (40). The mutant genes were sequenced and found to contain only the mutation desired.…”

Section: Methodsmentioning

confidence: 99%

Evolutionary Relationship between Different Subgroups of Restriction Endonucleases

Pingoud¹,

Кубарева²,

Stengel³

et al. 2002

The type II restriction endonuclease SsoII shows sequence similarity with 10 other restriction endonucleases, among them the type IIE restriction endonuclease EcoRII, which requires binding to an effector site for efficient DNA cleavage, and the type IIF restriction endonuclease NgoMIV, which is active as a homotetramer and cleaves DNA with two recognition sites in a concerted reaction. We show here that SsoII is an orthodox type II enzyme, which is active as a homodimer and does not require activation by binding to an effector site. Nevertheless, it shares with EcoRII and NgoMIV a very similar DNA-binding site and catalytic center as shown here by a mutational analysis, indicative of an evolutionary relationship between these three enzymes. We suggest that a similar relationship exists between other orthodox type II, type IIE, and type IIF restriction endonucleases. This may explain why similarities may be more pronounced between members of different subtypes of restriction enzymes than among the members of a given subtype.More than 3000 different type II restriction endonucleases are known and characterized with respect to their cleavage specificities (1). This group of enzymes probably constitutes one of the largest families of enzymes with the same basic function. This makes type II restriction endonucleases (like DNA methyltransferases) ideal objects to study evolutionary relationships. Moreover, in principle, the family relationships should eventually allow predicting structural and functional features of an individual restriction endonuclease, solely on the basis of sequence comparisons. This was not obvious for a long time since only 10 years ago restriction endonucleases, because of their little sequence conservation, were not considered to be related in evolution (2, 3). But this changed due to the progress made in the analysis of sequence similarities (4 -6) and, in particular, due to the increasing number of crystal structures of restriction enzymes (reviewed in Refs. 7 and 8), which made it obvious that these enzymes have very similar structural cores. Nevertheless, it has been shown recently that some genuine restriction enzymes belong to distinct nuclease superfamilies (Nuc, HNH, and GIY-YIG), which are unrelated and structurally dissimilar to each other and to the "archetypal" (P)D . . . (D/E)XK superfamily (9 -11). These findings make comparative studies using sequences of restriction enzymes even more challenging.We have begun to study the restriction endonuclease SsoII recently (12-15). It is a type II enzyme (reviewed in Refs. 8 and 16) composed of identical subunits each consisting of 305 amino acid residues (17). It cleaves the palindromic sequence 2CC-NGG in the presence of Mg 2ϩ as indicated (18). Our interest in studying this enzyme is due to its sequence similarity to Eco-RII, a type IIE enzyme (reviewed in Ref. 19), which being somewhat larger than SsoII is a dimer of identical subunits each consisting of 404 amino acid residues (20, 21). As a type IIE enzyme EcoRII has two DNA-binding...