Combination Of Library Screening Research Articles

A Combination of Library Screening and Rational Mutagenesis Expands the Available Color Palette of the Smallest Fluorogen-Activating Protein Tag nanoFAST.

NanoFAST is the smallest fluorogen-activating protein, consisting of only 98 amino acids, used as a genetically encoded fluorescent tag. Previously, only a single fluorogen with an orange color was revealed for this protein. In the present paper, using rational mutagenesis and in vitro screening of fluorogens libraries, we expanded the color palette of this tag. We discovered that E46Q is one of the key substitutions enabling the range of possible fluorogens to be expanded. The introduction of this and several other substitutions has made it possible to use not only orange but also red and green fluorogens with the modified protein.

Type I antifreeze proteins expressed in snailfish skin are identical to their plasma counterparts

Type I antifreeze proteins (AFPs) are usually small, Ala-rich alpha-helical polypeptides found in right-eyed flounders and certain species of sculpin. These proteins are divided into two distinct subclasses, liver type and skin type, which are encoded by separate gene families. Blood plasma from Atlantic (Liparis atlanticus) and dusky (Liparis gibbus) snailfish contain type I AFPs that are significantly larger than all previously described type I AFPs. In this study, full-length cDNA clones that encode snailfish type I AFPs expressed in skin tissues were generated using a combination of library screening and PCR-based methods. The skin clones, which lack both signal and pro-sequences, produce proteins that are identical to circulating plasma AFPs. Although all fish examined consistently express antifreeze mRNA in skin tissue, there is extreme individual variation in liver expression - an unusual phenomenon that has never been reported previously. Furthermore, genomic Southern blot analysis revealed that snailfish AFPs are products of multigene families that consist of up to 10 gene copies per genome. The 113-residue snailfish AFPs do not contain any obvious amino acid repeats or continuous hydrophobic face which typify the structure of most other type I AFPs. These structural differences might have implications for their ice-crystal binding properties. These results are the first to demonstrate a dual liver/skin role of identical type I AFP expression which may represent an evolutionary intermediate prior to divergence into distinct gene families.

Molecular recognition at purine and pyrimidine nucleotide (P2) receptors.

In comparison to other classes of cell surface receptors, the medicinal chemistry at P2X (ligand-gated ion channels) and P2Y (G protein-coupled) nucleotide receptors has been relatively slow to develop. Recent effort to design selective agonists and antagonists based on a combination of library screening, empirical modification of known ligands, and rational design have led to the introduction of potent antagonists of the P2X(1) (derivatives of pyridoxal phosphates and suramin), P2X(3)(A-317491), P2X(7) (derivatives of the isoquinoline KN-62), P2Y(1)(nucleotide analogues MRS 2179 and MRS 2279), P2Y(2)(thiouracil derivatives such as AR-C126313), and P2Y(12)(nucleotide/nucleoside analogues AR-C69931X and AZD6140) receptors. A variety of native agonist ligands (ATP, ADP, UTP, UDP, and UDP-glucose) are currently the subject of structural modification efforts to improve selectivity. MRS2365 is a selective agonist for P2Y(1)receptors. The dinucleotide INS 37217 potently activates the P2Y(2)receptor. UTP-gamma-S and UDP-beta-S are selective agonists for P2Y(2)/P2Y(4)and P2Y(6)receptors, respectively. The current knowledge of the structures of P2X and P2Y receptors, is derived mainly from mutagenesis studies. Site-directed mutagenesis has shown that ligand recognition in the human P2Y(1)receptor involves individual residues of both the TMs (3, 5, 6, and 7), as well as EL 2 and 3. The binding of the negatively-charged phosphate moiety is dependent on positively charged lysine and arginine residues near the exofacial side of TMs 3 and 7.

Cloning of a Na+-driven Cl/HCO3 exchanger from squid giant fiber lobe.

We extracted RNA from the giant fiber lobe (GFL) of the squid Loligo pealei and performed PCR with degenerate primers that were based on highly conserved regions of Na+-coupled HCO3- transporters. This approach yielded a novel, 290-bp sequence related to the bicarbonate transporter superfamily. Using an L. opalescens library, we extended the initial fragment in the 3' and 5' directions by a combination of library screening and PCR and obtained the full-length clone (1,198 amino acids) by PCR from L. pealei GFL. The amino acid sequence is 46% identical to mammalian electrogenic and electroneutral Na-HCO3 cotransporters and 33% identical to the anion exchanger AE1. Northern blot analysis showed strong signals in L. pealei GFL, optic lobe, and heart and weaker signals in gill and stellate ganglion. To assess function, we injected in vitro-transcribed cRNA into Xenopus oocytes and subsequently used microelectrodes to monitor intracellular pH (pHi) and membrane voltage (Vm). Superfusing these oocytes with 5% CO2-33 mM HCO3- caused a CO2-induced fall in pHi, followed by a slow recovery. The absence of a rapid HCO3- -induced hyperpolarization indicates that the pHi recovery mechanism is electroneutral. Ion substitutions showed that Na+ and Cl- are required on opposite sides of the membrane. Transport was blocked by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The characteristics of our novel clone fit those of a Na+-driven Cl/HCO3 exchanger (NDCBE).

Structure and juvenile hormone-mediated regulation of the HMG-CoA reductase gene from the Jeffrey pine beetle, Dendroctonus jeffreyi

In several pine bark beetle species, juvenile hormone (JH) III regulated 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-R) gene expression has an important role in monoterpenoid pheromone production in males. We investigated the structure and regulated expression of the HMG-R gene ( HMG-R) in the Jeffrey pine beetle, Dendroctonus jeffreyi. cDNA and genomic sequences were recovered using a combination of library screening and PCR. The transcribed portion of the gene spans 9.8 kb and is interrupted by 13 introns. When compared to vertebrate HMG-Rs, the distribution of intron sites suggests a functional role for those in the 5′ untranslated region and membrane anchor domains. Northern blots show that topically applied JH III stimulates HMG-R expression up to 30-fold in male D. jeffreyi, compared to untreated insects, in both a dose- and time-dependent manner. There was no increase in expression levels in similarly treated female insects. The expression pattern is consistent with the production of monoterpenoid pheromone components in male D. jeffreyi, and suggests the utility of the system as a new tool for studying the mechanism of JH action.

Molecular cloning and primary structure analysis of porcine pancreatic α-amylase

A cDNA library was constructed in a Uni-ZAP XR vector using mRNA isolated from porcine pancreas. A full-length α-amylase cDNA was obtained using a combination of library screening and nested polymerase chain reaction. Sequencing of the clone revealed a 1536-nucleotide (nt) open reading frame encoding a protein of 496 amino acid (aa) residues with a signal peptide of 15 aa. The calculated molecular mass of the enzyme was 55 354 Da, in accordance with those of the purified porcine pancreatic α-amylase forms (PPAI and PPAII) as determined by mass spectrometry. A comparison of the deduced aa sequence with published peptidic sequences of PPAI identified a number of mismatches. The sequence of the cDNA reported here provides a sequence reference for PPA in excellent agreement with the refined three-dimensional structures of both PPAI and PPAII. No evidence for a second variant was found in the cDNA library and it is most likely that PPAI and PPAII are two forms of the same protein. The primary structure of PPA shows high homology with human, mouse and rat pancreatic α-amylases. The 304–310 region, corresponding to a mobile loop involved in substrate binding and processing near the active site, is fully conserved.

Expression of the nuclear gene encoding mitochondrial ATP synthase subunit alpha in early development of Drosophila and sea urchin.

Complementary DNAs encoding nuclear-coded mitochondrial ATP synthase subunit alpha of Drosophila melanogaster and Strongylocentrotus purpuratus were obtained by a combination of library screening and redundant PCR. The entire coding sequence of the precursor polypeptide was inferred for both species. Southern blots to genomic DNA indicated that the gene is almost certainly single-copy in both organisms. Northern blots to RNA from staged developmental series showed that ATP synthase subunit alpha mRNA is represented in the egg, declines in abundance during cleavage, and is replenished by zygotic transcription in both species. However, the extent and timing of these changes differ significantly in the two species studied. Nuclear-coded and mitochondrially encoded ATP synthase genes appear to be temporally co-regulated in Drosophila, but not sea urchin development.

The molecular biology of the flavin-containing monooxygenases of man

cDNA clones encoding five distinct members of the FMO family of man (FMOs 1,2, 3, 4 and 5) were isolated by a combination of library screening and reverse transcription-polymerase chain reaction techniques. The deduced amino acid sequences of the human FMOs have 82–87% identity with their known orthologues in other mammals but only 51–57% similarity to each other. The hydropathy profiles of the proteins are very similar. From the calculated rate of evolution of FMOs (a 1% change in sequence per 6 million years) it would appear that individual members of the FMO gene family arose by duplication of a common ancestral gene some 250–300 million years ago. Each of the FMO genes was mapped by the polymerase chain reaction to the long arm of human chromosome 1. The localization of the FMO1 gene was further refined to 1q23–q25 by in situ hybridization of human metaphase chromosomes. RNase protection assays demonstrated that in man each FMO gene displays a distinct developmental and tissue-specific pattern of expression. In the adult, FMO1 is expressed in kidney but not in liver, whereas in the foetus its mRNA is abundant in both organs. FMO3 expression is essentially restricted to the liver in the adult and the mRNA is either absent, or present in low amounts, in foetal tissues. FMO4 is expressed more constitutively. Human FMO1 and FMO3 cDNAs were functionally expressed in prokaryotic and eukaryotic cells. FMO1 and FMO3, expressed in either system, displayed product stereoselectivity in their catalysis of the N-oxidation of the pro-chiral tertiary amines, N-ethyl- N-methylaniline (EMA) and pargyline. Both enzymes were stereoselective with respect to the production of the (−)- S-enantiomer of EMA N-oxide. But in the case of pargyline, the enzymes displayed opposite stereoselectivity, FMO1 producing solely the (+)-enantiomer and FMO3 predominantly the (−)-enantiomer of the N-oxide.

Cloning and regulation of the rat mdr2 gene.

We have cloned the complete cDNA encoding the rat mdr2 gene by a combination of library screening and the polymerase chain reaction. The sequence of rat mdr2 cDNA is highly similar to other members of the mdr gene family but the initiation of transcription, tissue distribution and regulation of expression of rat mdr2 diverge from the other isoforms. Primer extension analysis showed rat mdr2 mRNA to have a major transcription start point at -277 and a minor one at approximately -518. We constructed gene specific probes for rat mdr2 and mdr1b and compared the expression patterns of these two genes. The highest expression of mdr2 mRNA was in the muscle, heart, liver and spleen. Both mdr2 and 1b mRNA levels were elevated in the livers of rats treated with CCl4 or following partial hepatectomies although the time course of induction of each gene differed. Mdr1b increased by 12 to 24 hours while mdr2 did not increase until 48 hours. Treatment of isolated hepatocytes or RC3 cells with cycloheximide did not effect mdr2 mRNA. In contrast, mdr1b expression was increased. These data suggest that rat mdr2, unlike mdr1b, is not regulated by a negative trans-acting protein factor.