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PubMed2005

Functional analysis of transactivation by mutants of hepatitis B virus X gene in human hepatocellular carcinoma.

The X protein of the hepatitis B virus transactivates various cellular and viral promoters and enhancers. In this study, wild-type and mutants of the X gene, including the point mutations at codons 130 (AAG-->ATG, lysin-->methionine) and 131 (GTC-->ATC, valine-->isoleucine), commonly found in patients with human hepatocellular carcinoma (HCC), or deletions encompassing the same region, were cloned and inserted into expression vectors. Functional analysis of the mutants of the X gene was performed on the long terminal repeat of the Rous sarcoma virus in a transient transfection assay. A transactivating function was observed in the vector containing point mutations at codons 130 and 131 at the same level as that of wild-type X gene. Two constructs, each containing a different type of 8-nucleotide deletion mutant (codons 128-130 or 130-132,) dramatically lost their transactivating function. These findings suggest that the transactivating function is not necessarily associated with the development of HCC, and that not only transactivation by the X gene but also the mutation-enhanced oncogenic potential of the gene products could contribute to hepatocarcinogenesis.

Animals↗
PubMed2006

Calcium-sensing receptor dimerizes in the endoplasmic reticulum: biochemical and biophysical characterization of CASR mutants retained intracellularly.

Calcium-sensing receptor (CASR), expressed in parathyroid gland and kidney, is a critical regulator of extracellular calcium homeostasis. This G protein-coupled receptor exists at the plasma membrane as a homodimer, although it is unclear at which point in the biosynthetic pathway dimerization occurs. To address this issue, we have analyzed wild-type and mutant CASRs harboring R66H, R66C or N583X-inactivating mutations identified in familial hypocalciuric hypercalcemia/neonatal severe hyperparathyroid patients, which were transiently expressed in kidney cells. All mutants were deficient in cell signaling responses to extracellular CASR ligands relative to wild-type. All mutants, although as well expressed as wild-type, lacked mature glycosylation, indicating impaired trafficking from the endoplasmic reticulum (ER). Dimerized forms of wild-type, R66H and R66C mutants were present, but not of the N583X mutant. By immunofluorescence confocal microscopy of non-permeabilized cells, although cell surface expression was observed for the wild-type, little or none was seen for the mutants. In permeabilized cells, perinuclear staining was observed for both wild-type and mutants. By colocalization fluorescence confocal microscopy, the mutant CASRs were localized within the ER but not within the Golgi apparatus. By the use of photobleaching fluorescence resonance energy transfer microscopy, it was demonstrated that the wild-type, R66H and R66C mutants were dimerized in the ER, whereas the N583X mutant was not. Hence, constitutive CASR dimerization occurs in the ER and is likely to be necessary, but is not sufficient, for exit of the receptor from the ER and trafficking to the cell surface.

Biophysical Phenomena↗
PubMed1991

Site-directed mutagenesis of the m3 muscarinic receptor: identification of a series of threonine and tyrosine residues involved in agonist but not antagonist binding.

The hydrophobic core of all muscarinic receptors contains several conserved serine, threonine and tyrosine residues, most of which do not occur in any other G-protein coupled receptor. Since these amino acids can serve as potential hydrogen bond donors or acceptors, we have tested the hypothesis that they may be involved in the selective binding of muscarinic ligands. To eliminate the OH groups present in these residues, we have created nine single point mutations in the rat m3 muscarinic receptor by converting serine and threonine residues to alanine, and tyrosine residues to phenylalanine. The ligand binding and functional properties of these receptors were studied after transient expression in COS-7 cells. Six out of the nine mutant receptors (threonine and tyrosine mutations) showed strong reductions (approximately 10- to 40-fold lower than the wild-type receptor) in agonist binding affinities and reduced potencies in agonist-induced activation of phosphoinositide hydrolysis. Their antagonist binding properties, however, were similar to those of the wild-type m3 receptor. Despite their location on different transmembrane domains (III, V, VI and VII), all six mutations are positioned at a similar level (one to two helical turns away from the membrane surface) within the outer leaflet of the plasma membrane and may thus define the plain in which muscarinic agonists (but not antagonists) bind to their target receptor.

Acetylcholine↗
PubMedDate not supplied

Mutation in the neuronal voltage-gated sodium channel SCN1A in familial hemiplegic migraine.

BACKGROUND: Familial hemiplegic migraine is an autosomal dominant severe subtype of migraine with aura characterised by some degree of hemiparesis during the attacks. So far, mutations in two genes regulating ion translocation-CACNA1A and ATP1A2-have been identified in pedigrees with this disease. METHODS: To identify additional genes for familial hemiplegic migraine, we did a genome-wide linkage analysis of two disease pedigrees without mutations in CACNA1A and ATP1A2. Ion channel genes in the candidate interval were analysed for mutations, and the functional consequences of the recorded sequence alteration were determined. FINDINGS: We identified a novel locus for familial hemiplegic migraine on chromosome 2q24. Sequencing of candidate genes in this region revealed a heterozygous missense mutation (Gln1489Lys) in the neuronal voltage-gated sodium channel gene SCN1A, mutations of which have been associated with epilepsy. This same mutation was present in three families with familial hemiplegic migraine. It results in a charge-altering aminoacid exchange in the so-called hinged-lid domain of the protein, which is critical for fast inactivation of the channel. Whole-cell recordings in transiently transfected tsA201 cells expressing the highly homologous SCN5A sodium channel showed that the mutation induces a two-fold to four-fold accelerated recovery from fast inactivation without altering any of the other channel parameters investigated. INTERPRETATION: Dysfunction of the neuronal sodium channel SCN1A can cause familial hemiplegic migraine. Our findings have implications for the understanding of migraine aura. Moreover, our study reinforces the molecular links between migraine and epilepsy, two common paroxysmal disorders.

Adolescent↗
PubMed2003

Desensitization of capsaicin-activated currents in the vanilloid receptor TRPV1 is decreased by the cyclic AMP-dependent protein kinase pathway.

Proinflammatory prostaglandin E2 is known to sensitize sensory neurons to noxious stimuli. This sensitization is mediated by the cAMP-dependent protein kinase (PKA) signal pathway. The capsaicin receptor TRPV1, a non-selective cation channel of sensory neurons involved in the sensation of inflammatory pain, is a target of PKA-mediated phosphorylation. Our goal was to investigate the influence of PKA on Ca(2+)-dependent desensitization of capsaicin-activated currents. By using site-directed mutagenesis, we created point mutations at PKA consensus sites and studied wild-type and mutant channels transiently expressed in HEK293t cells under whole-cell voltage clamp. We found that forskolin, a stimulator of adenylate cyclase, decreased desensitization of TRPV1. The selective PKA inhibitor H89 inhibited this effect. Mimicking phosphorylation at PKA consensus sites by replacing Ser-6, Ser-116, Thr-144, Thr-370, Ser-502, Ser-774, or Ser-820 with aspartate resulted in five mutations (S116D, T144D, T370D, S774D, and S820D) that exhibited decreased desensitization as well. However, disrupting phosphorylation by replacing respective sites with alanine resulted in four mutations (S6A, T144A, T370A, and S820A) with desensitization properties resembling those of the aspartate mutations. Significant changes in relative permeabilities for Ca2+ over Na+ or in capsaicin sensitivity could not explain changes in desensitization properties of mutant channels. In mutations S116A, S116D, T370A, and T370D, pretreatment of cells with forskolin did not reduce desensitization as compared with wild-type and other mutant channels. We conclude that Ser-116 and possibly Thr-370 are the most important residues involved in the mechanism of PKA-dependent reduction of desensitization of capsaicin-activated currents.

Animals↗
PubMed2005

Comparison of the properties of rare variants of alpha1-proteinase inhibitor expressed in COS-1 cells and assessment of their potential as risk factors in human disease.

Among the more than 75 known variants of alpha(1)-proteinase inhibitor, a sub-population of rare, point mutations causing single amino acid replacements have been identified and classified as "at risk" alleles for development of pulmonary disease. In most cases, it is not clear how the amino acid replacements typical of these variants change the properties of the inhibitor to increase risk of disease in the affected individuals. To begin to address this question, we mutagenized a wild type alpha(1)-proteinase inhibitor cDNA to encode a panel of eight different point mutants reported to be associated with increased risk for development of pulmonary disease. These variants were then expressed in COS-l cells transiently transfected with plasmids containing the altered cDNAs. The effects of the mutations on the rates of secretion, cellular location, intracellular degradation, activity, stability, and tendency to aggregate were determined. Results of these studies show that, in some cases, the mutations affect the rate of secretion, the activity or both of these properties of alpha(1)-proteinase inhibitor in a manner consistent with its designation as an "at-risk" allele. In other cases, the mutations do not significantly change the properties of the inhibitor, suggesting that these may be normal variants and that their expression may not increase the risk of disease.

Amino Acid Substitution↗
PubMed1993

Transcription of the rat glucagon gene by the cyclic AMP response element-binding protein CREB is modulated by adjacent CREB-associated proteins.

The cyclic AMP (cAMP) response element (CRE) of the rat glucagon gene (Glu-CRE, 5'-TGACGTCA-3') mediates transcriptional responses to 8-bromo-cAMP and protein kinase A (PKA) in a glucagon-producing hamster islet cell line (InR1G9). By several different DNA-protein binding assays, we show that the transcription factor CREB binds to the CRE octamer and that additional nuclear proteins bind to sequences adjacent to the CRE. Mutation of the Glu-CRE octamer attenuates both the binding of CREB and cAMP-dependent PKA-stimulated transcriptional activity in transient transfection experiments but does not affect the binding of adjacent CREB-associated proteins. Progressive deletions and clustered point mutations of the sequences flanking the Glu-CRE identify sequences (5'-TCATT-3') located both 5' and 3' to the core CRE octamer that bind several proteins. Two proteins with molecular masses of 80 and 100 kDa bind to each of the 5' and 3' TCATT sites. Formation of additional protein-DNA complexes containing 45- and 20-kDa proteins depends upon the integrity of both TCATT sequences. Deletion or point mutation of the TCATT motif located on the 3' side of the CRE octamer results in enhanced transcriptional responses to PKA, suggesting that the CREB-associated proteins decrease the ability of CREB to mediate PKA-stimulated transcription. Results from these studies demonstrate that nucleotides flanking the core CRE octamer can influence the activity of the CRE by serving as binding sites for proteins that modulate the function of CREB and suggest a mechanism to explain why some consensus palindromic CREs are less responsive to cAMP stimulation than others.

Animals↗
PubMed1990

The Drosophila Shaker gene codes for a distinctive K+ current in a subset of neurons.

A transient K+ current coded by the Shaker gene was identified in muscle and expressed in Xenopus oocytes by injecting cRNA transcribed from a cloned cDNA. The Shaker current has not previously been identified in neurons. Mutational analysis now reveals that in neurons, Shaker is required for expression of a very rapidly inactivating K+ current with a depolarized steady-state inactivation curve. Together, these properties distinguish the Shaker-coded current from similar fast transient K+ currents coded by other genes. The Sh5 mutation further enhanced the depolarization of the Shaker current steady-state inactivation curve. Deletion of the Shaker gene completely removes the transient K+ current from a small percentage of neurons (15%) in a mixed population, and removes a portion of the whole-cell current in about 35% of neurons. The remaining 50% of neurons were apparently unaffected by deletion of the Shaker gene. The unique combination of rapid inactivation and depolarized steady-state inactivation of the Shaker current may reflect a unique functional role for this current in the nervous system such as the rapid repolarization of action potentials.

Action Potentials↗
PubMed1991

Mutations in the third alpha-helix of bovine growth hormone dramatically affect its intracellular distribution in vitro and growth enhancement in transgenic mice.

To investigate the relationship between the secondary structure of the third alpha-helix (amino acids 109-126) of bovine growth hormone (bGH) and the biological activity of the molecule, proline or glycine residues have been used as substitutes for native amino acids at positions 114, 118, 121, and 126, respectively. Mutations at the positions 114, 118, and 121 resulted in a dramatic decrease in bGH secretion by transiently transfected mouse L cells whereas the substitution of glycine for glutamate at position 126 (bGH-E126G) did not affect secretion. Immunofluorescence staining revealed that those nonsecretory bGH mutations possessed a different intracellular location as compared with wild-type bGH or the mutated secretory forms of bGH. Similar results were seen in the distribution of these mutated bGH molecules in transfected rat GH-3 cells. Transgenic mice that express wild-type bGH or bGH-E126G grew to approximately 1.6 times the mass of nontransgenic littermates. Transgenic mice that express two nonsecretory forms of mutated bGHs were found to lack the enhanced mouse growth phenotype in spite of elevated levels of serum bGH. These results suggest that the secondary structure in the third alpha-helix of bGH may be important for efficient intracellular targeting in vitro and in growth promotion in transgenic mice.

Animals↗
PubMed1990

Complementation of the rev gene mutation among human and simian lentiviruses.

The functional exchangeability of the rev gene was assessed in transient transfection experiments by using in vitro-constructed rev and gag mutants of the following three primate lentiviruses: human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus from the African green monkey (SIV AGM). Cotransfection into SW480 cells of the rev and gag mutants derived from the DNA of each infectious virus resulted in the generation of progeny particles as determined by reverse transcriptase assay. rev gene mutants of HIV-2 and SIV AGM were also complemented by all gag mutants derived from the three viruses. In contrast, no evidence of complementation was obtained following cotransfection of the HIV-1 rev mutant and the gag mutant of HIV-2 or SIV AGM.

Amino Acid Sequence↗
PubMed2005

Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion.

The SARS-coronavirus (SARS-CoV) is the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. To delineate functional domains of the SARS-CoV S glycoprotein, single point mutations, cluster-to-lysine and cluster-to-alanine mutations, as well as carboxyl-terminal truncations were investigated in transient expression experiments. Mutagenesis of either the coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted fusion peptide, or an adjacent but distinct region, severely compromised S-mediated cell-to-cell fusion, while intracellular transport and cell-surface expression were not adversely affected. Surprisingly, a carboxyl-terminal truncation of 17 amino acids substantially increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17 amino acids regulated the S fusogenic properties. In contrast, truncation of 26 or 39 amino acids eliminating either one or both of the two endodomain cysteine-rich motifs, respectively, inhibited cell fusion in comparison to the wild-type S. The 17 and 26 amino-acid deletions did not adversely affect S cell-surface expression, while the 39 amino-acid truncation inhibited S cell-surface expression suggesting that the membrane proximal cysteine-rich motif plays an essential role in S cell-surface expression. Mutagenesis of the acidic amino-acid cluster in the carboxyl terminus of the S glycoprotein as well as modification of a predicted phosphorylation site within the acidic cluster revealed that this amino-acid motif may play a functional role in the retention of S at cell surfaces. This genetic analysis reveals that the SARS-CoV S glycoprotein contains extracellular domains that regulate cell fusion as well as distinct endodomains that function in intracellular transport, cell-surface expression, and cell fusion.

Amino Acid Sequence↗
PubMed2007

Destabilization of transthyretin by pathogenic mutations in the DE loop.

Transthyretin single-amino-acid variants are responsible for familial amyloidotic polyneuropathy, in which transthyretin variants accumulate extracellularly in the form of fibrillar aggregates. We studied the structural stabilities of four transthyretin variants (L58H, L58R, T59K, and E61K), in which a positively charged amino acid is introduced in a loop region between the D- and E-strands. In addition to being located in the DE-loop, L58 and T59 are involved in the core of the transthyretin monomer. The L58H, L58R, and T59K substitutions destabilized transthyretin more than the E61K mutation did, indicating that transthyretin is substantially destabilized by the substitution of residues located in both the DE-loop and the monomer core. By utilizing hydrogen-deuterium exchange and nuclear magnetic resonance, we demonstrated that residues in the G-strand and the loop between the A- and B-strands were destabilized by these pathogenic mutations in the DE loop. At the quaternary structural level, the DE-loop mutations destabilized the dimer-dimer contact area, which may lead to transient dissociation into a dimer. Our results suggest that the destabilization of the dimer-dimer interface and the monomer core is important for the amyloidogenesis of transthyretin.

Amyloid↗
PubMed2001

Taf(II) 250 phosphorylates human transcription factor IIA on serine residues important for TBP binding and transcription activity.

Transcription factor IIA (TFIIA) is a positive acting general factor that contacts the TATA-binding protein (TBP) and mediates an activator-induced conformational change in the transcription factor IID (TFIID) complex. Previously, we have found that phosphorylation of yeast TFIIA stimulates TFIIA.TBP.TATA complex formation and transcription activation in vivo. We now show that human TFIIA is phosphorylated in vivo on serine residues that are partially conserved between yeast and human TFIIA large subunits. Alanine substitution mutation of serine residues 316 and 321 in TFIIA alphabeta reduced TFIIA phosphorylation significantly in vivo. Additional alanine substitutions at serines 280 and 281 reduced phosphorylation to undetectable levels. Mutation of all four serine residues reduced the ability of TFIIA to stimulate transcription in transient transfection assays with various activators and promoters, indicating that TFIIA phosphorylation is required globally for optimal function. In vitro, holo-TFIID and TBP-associated factor 250 (TAF(II)250) phosphorylated TFIIA on the beta subunit. Mutation of the four serines required for in vivo phosphorylation eliminated TFIID and TAF(II)250 phosphorylation in vitro. The NH(2)-terminal kinase domain of TAF(II)250 was sufficient for TFIIA phosphorylation, and this activity was inhibited by full-length retinoblastoma protein but not by a retinoblastoma protein mutant defective for TAF(II)250 interaction or tumor suppressor activity. TFIIA phosphorylation had little effect on the TFIIA.TBP.TATA complex in electrophoretic mobility shift assay. However, phosphorylation of TFIIA containing a gamma subunit Y65A mutation strongly stimulated TFIIA.TBP.TATA complex formation. TFIIA-gammaY65A is defective for binding to the beta-sheet domain of TBP identified in the crystal structure. These results suggest that TFIIA phosphorylation is important for strengthening the TFIIA.TBP contact or creating a second contact between TFIIA and TBP that was not visible in the crystal structure.

Amino Acid Sequence↗
PubMed1996

Translocation and transcriptional arrest during transcript elongation by RNA polymerase II.

RNA polymerase II may stop transcription, or arrest, while transcribing certain DNA sequences. The molecular basis for arrest is not well understood, but a connection has been suggested between arrest and a transient failure of the polymerase to translocate along the template. We have investigated this question by monitoring the movement of RNA polymerase II along a number of templates, using exonuclease III protection as our assay. We found that normal transcription is accompanied by essentially coordinate movement of the active site and both the leading and trailing edges of the polymerase. However, as polymerase approaches an arrest site, translocation of the body of the polymerase stops while transcription continues, leading to an arrested complex in which the 3' end of the transcript is located much closer than normal to the front edge of the polymerase. Surprisingly, mutated arrest sites that no longer block transcription continue to direct the transient failure of polymerase translocation. As transcription proceeds through these sequences, the initially stationary polymerase moves forward 10-15 bases along the template in response to the addition of only 3 bases to the nascent RNA. Mutagenesis studies indicate that the sequences responsible for the transient block to polymerase movement are located downstream of the T-rich motif required for arrest. Our results indicate that blocking translocation is not sufficient to cause arrest.

Base Composition↗
PubMed2006

An iterated sequence in the genome of Banana bunchy top virus is essential for efficient replication.

Banana bunchy top virus (BBTV) has a multi-component genome of circular, single-stranded DNA. BBTV replicates via a rolling-circle mechanism, probably involving sequence-specific interaction of the replication initiation protein (Rep) with iterated sequences (iterons) within the viral genome. Three putative iterons (designated F1, F2 and R), with the sequence GGGAC, have been identified in the intergenic region of each BBTV component. To investigate their role in replication, each of the iterons was mutated, singularly and in tandem, in a BBTV DNA-N 1.1mer and the ability of these molecules to be replicated by the BBTV 'master' Rep was evaluated in banana cells using transient biolistic assays. All iteron mutants were replicated less efficiently than the native DNA-N. Mutation of the F1 and R iterons caused a 42 and 62 % reduction in DNA-N replication, respectively, whereas mutation of the F2 and combined F1F2 iteron virtually abolished DNA-N replication.

Base Sequence↗
PubMed1994

The hormone-binding role of 2 cysteines near the C terminus of the mouse glucocorticoid receptor.

Previous biochemical analyses with covalent affinity labels and thiol-blocking reagents suggested possible roles for one methionine residue and multiple cysteine residues in binding of steroid to the 250-amino acid hormone-binding domain at the C-terminal end of mammalian glucocorticoid receptors. To test the functional roles of these residues in the receptor's ability to bind hormone and active transcription of target genes, the mouse glucocorticoid receptor cDNA was specifically mutated to cause single amino acid substitutions for methionine 610 and for each of the 5 cysteines (at positions 628, 644, 649, 671, and 742) in the hormone-binding domain. Among these 6 residues, only mutations in cysteine 671 and cysteine 742 caused substantial reductions in function. In transient transfection assays, the concentration of dexamethasone required for half-maximal activation of a glucocorticoid-responsive reporter gene was increased by 10-40-fold by changing cysteine 742 to serine or cysteine 671 to serine or alanine. At saturating concentrations of dexamethasone, the mutant receptors activated the reporter gene to the same extent as the wild type receptor, indicating that the mutations affected only the hormone-binding function of the receptor and not its ability to bind DNA or activate transcription once the hormone was bound.

Amino Acid Sequence↗
PubMed2001

Mutations of the C-terminal end of cathepsin K affect proenzyme secretion and intracellular maturation.

Transfection of the human cathepsin K cDNA into CHO cells results in the expression of mature catalytically active 27-kDa protein and in cells secreting the 39-kDa proenzyme form. Monensin, which neutralizes the pH of acidic organelles, was found to inhibit intracellular processing of the proenzyme and to stimulate its secretion into the culture medium. Brefeldin A caused alterations in immunofluorescence staining consistent with interference of lysosomal targeting and inhibited both intracellular processing and secretion of cathepsin K. Inhibition of glycosylation by tunicamycin also abolished cathepsin K maturation. Furthermore, the processing of the proenzyme to the mature form was abolished by a single mutation of the terminal Met(329) to Ala. The triple mutation of Ser(325), Pro(327), and Met(329) (all to Ala) inhibited both maturation and secretion, using either transient or stable expression systems. The results indicate that intracellular maturation and secretion of cathepsin K can be affected differentially by various treatments and by mutations of the C-terminal end of the protein. These results are consistent with the involvement of both the secreted proenzyme and the intracellularly processed enzyme in cathepsin K-mediated processes.

Acids↗
PubMed1994

Plasmid stability and ecological competence in recombinant cultures.

The instability of cell cultures containing plasmid vectors is a major problem in the commercial exploitation of molecular cloning techniques. Plasmid stability is influenced by the nature of the host cell, the type of plasmid and/or environmental conditions. Plasmid encoded properties may confer a selective advantage on the host cell but can be an energy drain due to replication and expression. Stability of recombinant cultures ultimately may be determined by the cost to benefit ratio of plasmid carriage. The relative competition between plasmid containing and plasmid-free or indigenous populations can determine the degree of dominance of recombinant cultures. The use of inocula in biotechnological processes in which dynamic environmental conditions dominate may also result in instabilities resulting from the characteristics of the ecosystem. In such dynamic conditions plasmid stability is just one contribution to culture stability. Strategies to enhance plasmid stability, within such environments, based on manipulation of physiological state of host cells, must consider the responsiveness or plasticity of both cells and populations. The robustness of cells or the responses to stresses or transient environmental conditions can influence the levels of instability detected; for example, instability or mutation in the host genome may lead to enhanced plasmid stability. Competition among subpopulations arising from unstable copy number control may determine the levels of recombinant cells in open versus closed fermenter systems. Thus the ecological competence (ability to survive and compete) of recombinant cells in dynamic or transient environments is fundamental to the understanding of the ultimate dominance or survival of such recombinant cultures and may form the basis of a strategy to enhance or control stability either in fermenter systems or dynamic process environments. The creation of microniches in time and/or space can enhance plasmid stability. Transient operation based on defined environmental stresses or perturbations in fermenter systems or in heterogeneous or dynamic environments found in gel immobilized cultures have resulted in enhanced stability. Spatial organization resulting from immobilization has the additional advantage of regulated cell protection within defined microenvironments and controlled release, depending on the nature of the gel, from these microenvironments or microcosms. This regulation of ecological competence allied to the advantages of microbial cell growth in gel microenvironments combined with the spatial organization (or juxtapositioning of cells, selective agents, nutrients, protectants, etc.) possible through immobilization technology offers new strategies to enhance plasmid and culture stability.

Journal Article↗