Unique Restriction Enzyme Research Articles

A convenient plasmid system for construction of helper‐dependent adenoviral vectors and its application for analysis of the breast‐cancer‐specific mammaglobin promoter

Helper-dependent (HD) adenovirus (Ad) vectors, deleted of all viral coding sequences, have a higher cloning capacity, improved performance of tissue-specific promoters, and reduced toxicity in animals relative to first-generation Ad vectors, making these vectors promising tools for gene transfer in vitro and in vivo. However, the large size of HDAd precursor plasmids renders them relatively difficult to manipulate due to the paucity of unique restriction enzyme sites suitable for transgene insertion and to the size constraints imposed by the viral packaging machinery. We have constructed a series of HDAd precursor plasmids that allows cassette insertion at a unique site in the vector backbone. We have tested whether these vector backbones will support the tissue-specificity of inserted expression cassettes in a study of the activity of the potentially breast-cancer-specific mammaglobin promoter and enhancer. We report here the generation of a series of HDAd precursor plasmids, both with and without an additional reporter expression cassette, that were designed to accommodate a wide range in size of inserted DNA. The system was validated for transcriptional targeting studies by demonstrating the tissue-specificity and activity of the mammaglobin promoter rescued using this precursor system. In addition, we have extended our previous studies on the mammaglobin promoter by demonstrating that two copies of the mammaglobin enhancer fused to the minimal promoter surpassed the activity of the single enhancer/promoter by at least 10-fold in breast cancer cells while maintaining only minimal expression in normal cells both in vitro and in a mouse tumor model. This versatile plasmid system simplifies the construction of HDAd vectors and was valuable in demonstrating the targeting potential of the mammaglobin promoter for breast cancer gene therapy.

Ecology of Escherichia coli O157:H7 in Commercial Dairies in Southern Alberta

Shedding of Escherichia coli O157:H7 was monitored monthly over a 1-yr period by collecting pooled fecal pats (FECAL) and manila ropes orally accessed for 4h (ROPE) from multiple pens of cattle in 5 commercial dairies in southern Alberta, Canada. Using immunomagnetic separation, E. coli O157:H7 was isolated from cows on 4 of the dairies and from 13.5% of FECAL and 1.1% of ROPE samples. Pulsed-field gel electrophoresis of XbaI- and SpeI-digested bacterial DNA of the 65 isolates produced 23 unique restriction endonuclease digestion patterns, although 92% of the isolates belonged to 3 restriction endonuclease digestion pattern clusters sharing a minimum 90% homology. Collection of positive isolates was 15 times more likely from June through September. Across dairies, peak somatic cell count occurred in July, August, September, and November. The likelihood of positive isolates was 2.6 times higher in calves and heifers compared with mature cows. This study indicates that ROPE would be of little value for the detection of E. coli O157:H7 in dairy herds unless oral contact with ROPE could be increased in mature animals. Additionally, mitigation strategies for E. coli O157:H7 should be targeted to the months of July, August, and September and toward immature animals for maximum impact. All farms displayed unique combinations of seasonality of shedding and diversity of E. coli O157:H7 subtypes. The fact that seasonal prevalence of E. coli O157:H7 largely coincided with peak somatic cell count within climatically controlled dairy barns suggests that similar environmental factors may be enhancing fecal shedding E. coli O157:H7 and the incidence of mastitis.

“Natively Unfolded” VPg Is Essential for Sesbania Mosaic Virus Serine Protease Activity

Polyprotein processing is a major strategy used by many plant and animal viruses to maximize the number of protein products obtainable from a single open reading frame. In Sesbania mosaic virus, open reading frame-2 codes for a polyprotein that is cleaved into different functional proteins in cis by the N-terminal serine protease domain. The soluble protease domain lacking 70-amino-acid residues from the N terminus (deltaN70Pro, where Pro is protease) was not active in trans. Interestingly, the protease domain exhibited trans-catalytic activity when VPg (viral protein genome-linked) was present at the C terminus. Bioinformatic analysis of VPg primary structure suggested that it could be a disordered protein. Biophysical studies validated this observation, and VPg resembled "natively unfolded" proteins. CD spectral analysis showed that the deltaN70Pro-VPg fusion protein had a characteristic secondary structure with a 230 nm positive CD peak. Mutation of Trp-43 in the VPg domain to phenylalanine abrogated the positive peak with concomitant loss in cis- and trans-proteolytic activity of the deltaN70Pro domain. Further, deletion of VPg domain from the polyprotein completely abolished proteolytic processing. The results suggested a novel mechanism of activation of the protease, wherein the interaction between the natively unfolded VPg and the protease domains via aromatic amino acid residues alters the conformation of the individual domains and the active site of the protease. Thus, VPg is an activator of protease in Sesbania mosaic virus, and probably by this mechanism, the polyprotein processing could be regulated in planta.

Proximity of Transmembrane Domains 1 and 3 of the γ-AminobutyricAcid Transporter GAT-1 Inferred from Paired CysteineMutagenesis

GAT-1 is a sodium- and chloride-dependent gamma-aminobutyric acid transporter and is the first identified member of a family of transporters that maintain low synaptic neurotransmitter levels and thereby enable efficient synaptic transmission. Because transmembrane domains 1 and 3 contain amino acid residues important for transport activity, we hypothesized that these domains may participate in the formation of the binding pocket of the transporter. Pairwise substitutions have been introduced in several predicted transmembrane domains and in the first extracellular loop of GAT-1. In the double mutant W68C/I143C, in which the cysteines were introduced at locations at the extracellular part of transmembrane domains 1 and 3, respectively, approximately 70% inhibition of transport was observed by cadmium with an IC50 of approximately 10 microm. This inhibition was not observed in the corresponding single mutants and also not in > 10 other double mutants, except for V67C/I143C, where the half-maximal effect was obtained at approximately 50 microm. The inhibition by cadmium was only observed when the cysteine pairs were introduced in the same polypeptide. Our results suggest that transmembrane domains 1 and 3 come in close proximity within the transporter monomer.

Dual Functionality of the Anti-β1 Integrin Antibody, 12G10, Exemplifies Agonistic Signalling from the Ligand Binding Pocket of Integrin Adhesion Receptors

Although integrins are known to mediate connections between extracellular adhesion molecules and the intracellular actin cytoskeleton, the mechanisms that are responsible for coupling ligand binding to intracellular signaling, for generating diversity in signaling, and for determining the efficacy of integrin signaling in response to ligand engagement are largely unknown. By characterizing the class of anti-integrin monoclonal antibodies (mAbs) that stimulate integrin activation and ligand binding, we have identified integrin-ligand-mAb complexes that exhibit differential signaling properties. Specifically, addition of 12G10 mAb to cells adhering via integrin alpha4beta1 was found to trigger disruption of the actin cytoskeleton and prevent cell attachment and spreading, whereas mAb addition to cells adhering via alpha5beta1 stimulated all of these processes. In contrast, soluble ligand binding to either alpha4beta1 or alpha5beta1 was augmented or unaffected by 12G10. The regions of the integrin responsible for differential signaling were then mapped using chimeras. Surprisingly, a chimeric alpha5 integrin containing the beta-propeller domain from the ligand binding pocket of alpha4 exhibited the same signaling properties as the full-length alpha4 integrin, whereas exchanging or removing cytoplasmic domains had no effect. Thus the mAb 12G10 demonstrates dual functionality, inhibiting cell adhesion and spreading while augmenting soluble ligand binding, via a mechanism that is determined by the extracellular beta-propeller domain of the associating alpha-subunit. These findings therefore demonstrate a direct and variable agonistic link between the ligand binding pocket of integrins and the cell interior that is independent of the alpha cytoplasmic domains. We propose that either ligand-specific transmembrane conformational changes or ligand-specific differences in the kinetics of transmembrane domain separation underlie integrin agonism.

Simultaneous Inhibition of Epidermal Growth Factor Receptor (EGFR) Signaling and Enhanced Activation of Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Receptor-mediated Apoptosis Induction by an scFv:sTRAIL Fusion Protein with Specificity for Human EGFR

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent with tumor-selective apoptotic activity. Here we present a novel approach that combines EGFR-signaling inhibition with target cell-restricted apoptosis induction using a TRAIL fusion protein with engineered specificity for EGFR. This fusion protein, scFv425:sTRAIL, comprises the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL (sTRAIL). Treatment with scFv425:sTRAIL resulted in the specific accretion to the cell surface of EGFR-positive cells only. EGFR-specific binding rapidly induced a dephosphorylation of EGFR and down-stream mitogenic signaling, which was accompanied by cFLIP(L) down-regulation and Bad dephosphorylation. EGFR-specific binding converted soluble scFv425:sTRAIL into a membrane-bound form of TRAIL that cross-linked agonistic TRAIL receptors in a paracrine manner, resulting in potent apoptosis induction in a series of EGFR-positive tumor cell lines. Co-treatment of EGFR-positive tumor cells with the EGFR-tyrosine kinase inhibitor Iressa resulted in a potent synergistic pro-apoptotic effect, caused by the specific down-regulation of c-FLIP. Furthermore, in mixed culture experiments binding (L)of scFv425:sTRAIL to EGFR-positive target cells conveyed a potent apoptotic effect toward EGFR-negative bystander tumor cells. The favorable characteristics of scFv425:sTRAIL, alone and in combination with Iressa, as well as its potent anti-tumor bystander activity indicate its potential value for treatment of EGFR-expressing cancers.

The High Resolution Crystal Structure of a Native Thermostable Serpin Reveals the Complex Mechanism Underpinning the Stressed to Relaxed Transition

Serpins fold into a native metastable state and utilize a complex conformational change to inhibit target proteases. An undesirable result of this conformational flexibility is that most inhibitory serpins are heat sensitive, forming inactive polymers at elevated temperatures. However, the prokaryote serpin, thermopin, from Thermobifida fusca is able to function in a heated environment. We have determined the 1.8 A x-ray crystal structure of thermopin in the native, inhibitory conformation. A structural comparison with the previously determined 1.5 A structure of cleaved thermopin provides detailed insight into the complex mechanism of conformational change in serpins. Flexibility in the shutter region and electrostatic interactions at the top of the A beta-sheet (the breach) involving the C-terminal tail, a unique structural feature of thermopin, are postulated to be important for controlling inhibitory activity and triggering conformational change, respectively, in the native state. Here we have discussed the structural basis of how this serpin reconciles the thermodynamic instability necessary for function with the stability required to withstand elevated temperatures.

Improvement of the bioluminescence reporter system for real-time monitoring of circadian rhythms in the cyanobacterium Synechocystis sp. strain PCC 6803

Circadian rhythm is a self-sustaining oscillation whose period length coincides with the 24-hour day-night cycle. A powerful tool for circadian clock research is the real-time automated bioluminescence monitoring system in which a promoter region of a clock-controlled gene is fused to a luciferase reporter gene and rhythmic regulation of the promoter activity is monitored as bioluminescence. In the present study, we greatly improved the bioluminescence reporter system in the cyanobacterium Synechocystis sp. strain PCC 6803. We fused an 805-bp promoter region of the dnaK gene seamlessly to the luxA coding sequence and integrated the P(dnaK)::luxAB fusion gene into a specific intergenic region of the Synechocystis genome (targeting site 1). The resulting new reporter strain, PdnaK::luxAB(-), showed 12 times the bioluminescence intensity of the standard reporter strain, CFC2. Furthermore, we generated strain PdnaK::luxAB(+), in which the P(dnaK)::luxAB fusion gene and the selection-marker spectinomycin resistance gene are transcribed in opposite directions. The PdnaK::luxAB(+) strain showed 19 times the bioluminescence intensity of strain CFC2. The procedures used to increase the bioluminescence intensity are especially useful for bioluminescence monitoring of genes with low promoter activity. In addition, these reporter constructs facilitate bioluminescence monitoring of any gene because the promoter fragments they contain can easily be replaced by digestion with unique restriction enzymes. They would therefore contribute to a genome-wide analysis of gene expression in Synechocystis.

124 KNOCKING-IN OF A FOREIGN GENE ON A BOVINE BETA-CASEIN GENE INTO BOVINE PRIMARY FIBROBLASTS USING HOMOLOGOUS RECOMBINATION EVENTS

Knocking-in of a foreign gene on a tissue-specific genomic region has not been reported in livestock. In this study, we constructed two knock-in vector cassettes specific for the bovine beta-casein gene which is expressed only in the mammary gland during lactation. The targeting vector cassettes, pBCKI1 and pBCKI2, have homology regions for the bovine beta-casein gene and contain 13.1 kb and 9.1 kb targeting arms with different long arm lengths, respectively. The targeting vector cassettes have unique restriction enzyme sites for insertion of foreign therapeutic genes in front of the neo gene which was inserted into the vector as a selection marker. The human thrombopoietin (hTPO) gene was inserted into the restriction enzyme sites of both targeting vectors, which were named pBCTPOKI1 and pBCTPOKI2. When the two targeting vectors were transfected into bovine ear skin fibroblasts using Lipofectamine (Invitrogen, Seoul, South Korea), 6.3% of neo resistant clones (2/32) were homologously targeted with the pBCTPOKI2 vector. Cells from the targeted colonies were nuclear-transferred into enucleated bovine oocytes and cultured to the blastocyst stage. A total of 66 blastocysts were generated of which 46 were transferred into recipients. No offspring have been produced at this time. Here we first describe knock-in vectors specific for a bovine beta-casein gene, which will be employed to generate animal bioreactors that produce therapeutic proteins secreted into the milk.

109. Cyclical Mechanical Stretch and AAV2 Mediated Gene Transfer to Cultured Human Vascular Cells

Safe and durable transgene expression for the treatment of hepatic diseases has been reported utilizing several different viral vector systems. Following gene transfer, over-expression of some gene products may be undesirable. To this end, we prepared a recombinant AAV reporter vector that would be susceptible to molecular recombination that would terminate transgene expression. The AAV beta-galactosidase (LacZ) reporter vector (pAAV-LacZ) was modified to include loxP sites at two unique restriction enzyme sites flanking the LacZ expression cassette (pAAV-loxPLacZ). Sequential treatment of cultured human hepatoma cells (SKHep1) transfected with pAAV-loxPLacZ, followed by infection with a first generation adenovirus expressing the Cre recombinase (Ad/Cre) resulted in a 67% reduction of LacZ expression compared to control cells. Additional studies towards optimization of this molecular recombination as a form of reversible transgene expression are currently in progress. These in vitro studies represent an important step in the development of a safe, durable and reversible AAV-gene delivery system.

Molecular Cloning of a Sixth Member of the K+-dependent Na+/Ca2+ Exchanger Gene Family, NCKX6

Bioinformatic and molecular cloning tools were used to identify and isolate cDNA clones from mouse and human tissues that encode the sixth member of the K(+)-dependent Na+/Ca2+ exchanger family, NCKX6. The mouse NCKX6 protein is 585 amino acids long and shares about 62% sequence similarity with previously identified exchangers in the alpha-repeat regions but has little primary sequence similarity outside these regions. NCKX6 transcripts of 4 kb are abundantly expressed in all tissues examined and are thus more broadly distributed than previously described NC(K)X family members. Two alternatively spliced products of this novel gene were identified that encode proteins of different length. The short isoform differs from the full-length isoform at the C-terminal hydrophobic domain as a result of a shift in the reading frame caused by the deletion of two exons. Both NCKX6 isoforms were expressed in HEK-293 cells. Functional analysis by digital imaging of fura-2 loaded transfected HEK-293 cells demonstrated that the short isoform exhibited K(+)-dependent Na+/Ca2+ exchange activity whereas the full-length isoform did not. The latter was retained within the endoplasmic reticulum, whereas the short isoform was present at the plasma membrane in transfected cells. Immunofluorescence studies examining NCKX6 expression in native tissue using an NCKX6-specific antibody showed intense labeling of the cardiac sarcolemmal membrane. The discovery of NCKX6 therefore reveals a novel member of the Na+/Ca2+ exchanger superfamily whose ubiquitous expression in all tissues suggests an important role for K(+)-dependent Na+/Ca2+ exchange in maintaining cellular Ca2+ homeostasis in diverse tissues and cell types.

Longitudinal study of fecal shedding of Escherichia coli O157:H7 in feedlot cattle: predominance and persistence of specific clonal types despite massive cattle population turnover.

Identification of the sources and methods of transmission of Escherichia coli O157:H7 in feedlot cattle may facilitate the development of on-farm control measures for this important food-borne pathogen. The prevalence of E. coli O157:H7 in fecal samples of commercial feedlot cattle in 20 feedlot pens between April and September 2000 was determined throughout the finishing feeding period prior to slaughter. Using immunomagnetic separation, E. coli O157:H7 was isolated from 636 of 4,790 (13%) fecal samples in this study, with highest prevalence earliest in the feeding period. No differences were observed in the fecal or water trough sediment prevalence values of E. coli O157:H7 in 10 pens supplied with chlorinated drinking water supplies compared with nonchlorinated water pens. Pulsed-field gel electrophoresis of XbaI-digested bacterial DNA of the 230 isolates obtained from eight of the pens revealed 56 unique restriction endonuclease digestion patterns (REDPs), although nearly 60% of the isolates belonged to a group of four closely related genetic subtypes that were present in each of the pens and throughout the sampling period. The other REDPs were typically transiently detected, often in single pens and on single sample dates, and in many cases were also closely related to the four predominant REDPs. The persistence and predominance of a few REDPs observed over the entire feeding period on this livestock operation highlight the importance of the farm environment, and not necessarily the incoming cattle, as a potential source or reservoir of E. coli O157:H7 on farms.

The Interaction of the γ-Aminobutyric Acid Transporter GAT-1 with the Neurotransmitter Is Selectively Impaired by Sulfhydryl Modification of a Conformationally Sensitive Cysteine Residue Engineered into Extracellular Loop IV

The (Na+ + Cl-)-coupled gamma-aminobutyric acid (GABA) transporter GAT-1 keeps synaptic levels of this neurotransmitter low and thereby enables efficient GABA-ergic transmission. Extracellular loops (III, IV, and V) have been shown to contain determinants for GABA selectivity and affinity. Here we analyze the role of extracellular loop IV in transport by cysteine scanning mutagenesis. Fourteen residues of this loop have been replaced by cysteine. GABA transport by eight of the fourteen mutants is markedly more sensitive to inhibition by membrane-impermeant methane thiosulfate reagents than wild-type. Mutant A364C has high activity and is potently inhibited by the sulfhydryl reagent. GABA transport by the A364C/C74A double mutant, where the only externally accessible cysteine residue of the wild-type has been replaced by alanine, is also highly sensitive to the sulfhydryl reagents. Maximal sensitivity is observed in the presence of the cosubstrates sodium and chloride. A marked protection is afforded by GABA, provided sodium is present. This protection is also observed at 4 degrees C. The non-transportable analogue SKF100330A also protects the double mutant against sulfhydryl modification in the presence of sodium but has the opposite effect in its absence. Electrophysiological analysis shows that upon sulfhydryl modification of this mutant, GABA can no longer induce transport currents. The voltage dependence of the transient currents indicates an increased apparent affinity for sodium. Moreover, GABA is unable to suppress the transient currents. Our results indicate that part of extracellular loop IV is conformationally sensitive, and its modification selectively abolishes the interaction of the transporter with GABA.

Species identification of Hypoderma affecting domestic and wild ruminants by morphological and molecular characterization.

Cuticular structures and the sequence of the cytochrome oxidase I gene were compared for Hypoderma bovis (Linnaeus), Hypoderma lineatum (De Villers), Hypoderma actaeon Brauer, Hypoderma diana Brauer and Hypoderma tarandi (Linnaeus) (Diptera, Oestridae). Third-stage larvae of each species were examined by scanning electron microscopy revealing differences among species in the pattern and morphology of spines on the cephalic and thoracic segments, by spine patterns on the tenth abdominal segment, and by morphology of the spiracular plates. The morphological approach was supported by the molecular characterization of the most variable region of the cytochrome oxidase I (COI) gene of these species, which was amplified by polymerase chain reaction and analysed. Amplicons were digested with the unique restriction enzyme, BfaI, providing diagnostic profiles able to simultaneously differentiate all Hypoderma species examined. These findings confirm the utility of morphological characters for differentiating the most common Hypoderma larvae and reconfirm the power of the COI gene for studying insect identification and systematics.

Characterization of a Polymerase Chain Reaction–Based Approach for the Simultaneous Detection of Multiple Animal-Derived Materials in Animal Feed

In this study, a polymerase chain reaction (PCR) primer set capable of amplifying a mitochondrial DNA segment of multiple species (cattle, sheep, goats, deer, and elk) whose rendered remains are prohibited from being fed to ruminants was characterized. However, the primer set also amplifies DNA derived from the rendered remains of pigs and horses, which are exempt from the feed ban. PCR amplicons derived from pig DNA have a restriction endonuclease site recognized by Hinf1, while the horse DNA–derived amplicon has a unique restriction endonuclease site recognized by HypCH4III. This “universal” PCR primer produced an amplicon with DNA extracted from dairy feed containing either bovine meat and bone meal or pig blood meal. Enzymatic digestion of the PCR amplicons from these feed samples with Hinf1 resulted in cleavage products only from samples containing pig blood meal. However, Hinf1 digestion of these amplicons was not complete. Further analysis of the pig blood meal with primers specific for bovine or porcine DNA demonstrated the presence of both bovine- and porcine-derived DNA. Enzymatic digestion confirmed these findings. Additional testing was conducted with dry dog food samples labeled as containing either lamb, chicken, turkey, or chicken and fish. The universal PCR primer produced an amplicon only for the dog food containing lamb meal. This paper is the first to describe a simplified approach for the detection of the prohibited species of concern in the feed ban.

Mutations in the genes for synthesis of the outer core region of the lipopolysaccharide of Yersinia enterocolitica O:3.

The aim of this study was to construct non-polar frame-shift mutations in some of the individual genes responsible for the biosynthesis of the branching outer core (OC) hexasaccharide of the lipopolysaccharide (LPS) in Yersinia enterocolitica O:3 (YeO:3). Chromosomal segments of YeO:3 containing wbcN, wbcO and wbcQ genes were cloned into a suicide vector. A frame-shift mutation was introduced into each gene by modifying a unique restriction enzyme recognition site. Each recombinant plasmid with a modified OC gene was mobilized into YeO:3 to allow for allelic exchange between the modified gene and the wild type chromosomal gene. The exchange was confirmed by demonstrating the absence of the particular restriction site in the chromosome of each mutant strain. Analysis of LPS by gel electrophoresis showed that the LPS of the mutants was lacking the OC. Therefore, the constructed wbcN, wbcO and wbcQ strains are true mutants with frame-shifts in the corresponding genes. The products of the wbcN, wbcO and wbcQ genes are putative glycosyltransferases and, based on the present analysis, essential for the biosynthesis of the OC hexasaccharide. The absence of OC in the LPS of these mutants further supports the hypothesis that the OC hexasaccharide is a single O-antigen O-unit that is not polymerized in YeO:3. These mutants provide information on the unique nature of the synthesis of OC of YeO:3 LPS. They are valuable for future biochemical studies to establish the roles of the products of individual OC genes.

Transmissible gastroenteritis coronavirus gene 7 is not essential but influences in vivo virus replication and virulence

Transmissible gastroenteritis coronavirus (TGEV) contains eight overlapping genes that are expressed from a 3′-coterminal nested set of leader-containing mRNAs. To facilitate the genetic manipulation of the viral genome, genes were separated by duplication of transcription regulating sequences (TRSs) and introduction of unique restriction endonuclease sites at the 5′ end of each gene using an infectious cDNA clone. The recombinant TGEV (rTGEV) replicated in cell culture with similar efficiency to the wild-type virus and stably maintained the modifications introduced into the genome. In contrast, the rTGEV replication level in the lungs and gut of infected piglets and virulence were significantly reduced. rTGEV in which gene 7 expression was abrogated (rTGEV-Δ7) were recovered from cDNA constructs, indicating that TGEV gene 7 was a nonessential gene for virus replication. Interestingly, in vivo infections with rTGEV-Δ7 showed an additional reduction in virus replication in the lung and gut, and in virulence, indicating that TGEV gene 7 influences virus pathogenesis.

Optimized bacterial expression of human apolipoprotein A-I

Apolipoprotein A-I (apoA-I) serves critical functions in plasma lipoprotein metabolism as a structural component of high density lipoprotein, activator of lecithin:cholesterol acyltransferase, and acceptor of cellular cholesterol as part of the reverse cholesterol transport pathway. In an effort to facilitate structure:function studies of human apoA-I, we have optimized a plasmid vector for production of recombinant wild type (WT) and mutant apoA-I in bacteria. To facilitate mutagenesis studies, subcloning, and DNA manipulation, numerous silent mutations have been introduced into the apoA-I cDNA, generating 13 unique restriction endonuclease sites. The coding sequence for human apoA-I has been modified by the introduction of additional silent mutations that eliminate 18 separate codons that employ tRNAs that are of low or moderate abundance in Escherichia coli. Yields of recombinant apoA-I achieved using the optimized cDNA were 100±20 mg/L bacterial culture, more than fivefold greater than yields routinely obtained with the original cDNA. Site-directed mutagenesis of the apoA-I cDNA was performed to generate a Glu2Asp mutation in the N-terminal sequence of apoA-I. This modification, which creates an acid labile Asp–Pro peptide bond between amino acids 2 and 3, permits specific chemical cleavage of an N-terminal His-Tag fusion peptide used for rapid protein purification. The product protein’s primary structure is identical to WT apoA-I in all other respects. Together, these changes in apoA-I cDNA and bacterial expression protocol significantly improve the yield of apoA-I protein without compromising the relative ease of purification.

Pulsed-Field Fingerprinting of Vaginal Group B Streptococcus in Pregnancy

In Brief OBJECTIVE There is more to be learned about the epidemiology of group B β-hemolytic streptococci infections in pregnancy. In this study, we investigated the discriminating capabilities of pulsed-field gel electrophoresis of group B streptococci strains from pregnant patients and mother/infant pairs of patients compared with serotyping. METHODS Forty-two vaginal strains of group B streptococci cultured from pregnant patients in the third trimester and strains from 20 mother/infant pairs with documented newborn group B streptococci infection were studied. Isolates were serotyped by the Lancefield capillary precipitin method and molecularly characterized by counterclamped homogeneous electrical field pulsed-field gel electrophoresis with rarely cutting restriction enzymes. RESULTS Nine of the 13 serotypes of group B streptococci identified thus far in the scientific literature (Ia, Ia/c, Ib, Ib/c, II, IIc, III, V, and NT/c) were represented among the 62 isolates. Among the 42 maternal isolates, eight serotypes were represented, and among the 20 mother/infant isolates, six serotypes were represented. Serotypes of mother/infant isolates matched in nine of the ten pairs. Restriction endonuclease profiles, or digests, from the 42 maternal isolates resulted in 25 unique profiles that were arranged into five major groups based on the overall relatedness. Each group was comprised of one predominant serotype. The 20 mother/infant paired isolates displayed nine unique restriction endonuclease profiles and nine of the ten paired isolates showed indistinguishable restriction endonuclease profiles between mother and infant. CONCLUSION Deoxyribonucleic acid profiling using pulsed-field gel electrophoresis is more discriminating of group B streptococci strains than serotyping because of the different yet closely related patterns within each restriction endonuclease profile group that are linked to one specific serotype. Pulsed-field gel electrophoresis can refine our epidemiologic studies of group B streptococci transmission and acquisition. Pulsed-field gel electrophoresis of group B streptococci is more discriminating than serotyping.

Pulsed-field fingerprinting of vaginal group B streptococcus in pregnancy

OBJECTIVE: There is more to be learned about the epidemiology of group B β-hemolytic streptococci infections in pregnancy. In this study, we investigated the discriminating capabilities of pulsed-field gel electrophoresis of group B streptococci strains from pregnant patients and mother/infant pairs of patients compared with serotyping. METHODS: Forty-two vaginal strains of group B streptococci cultured from pregnant patients in the third trimester and strains from 20 mother/infant pairs with documented newborn group B streptococci infection were studied. Isolates were serotyped by the Lancefield capillary precipitin method and molecularly characterized by counterclamped homogeneous electrical field pulsed-field gel electrophoresis with rarely cutting restriction enzymes. RESULTS: Nine of the 13 serotypes of group B streptococci identified thus far in the scientific literature (Ia, Ia/c, Ib, Ib/c, II, IIc, III, V, and NT/c) were represented among the 62 isolates. Among the 42 maternal isolates, eight serotypes were represented, and among the 20 mother/infant isolates, six serotypes were represented. Serotypes of mother/infant isolates matched in nine of the ten pairs. Restriction endonuclease profiles, or digests, from the 42 maternal isolates resulted in 25 unique profiles that were arranged into five major groups based on the overall relatedness. Each group was comprised of one predominant serotype. The 20 mother/infant paired isolates displayed nine unique restriction endonuclease profiles and nine of the ten paired isolates showed indistinguishable restriction endonuclease profiles between mother and infant. CONCLUSION: Deoxyribonucleic acid profiling using pulsed-field gel electrophoresis is more discriminating of group B streptococci strains than serotyping because of the different yet closely related patterns within each restriction endonuclease profile group that are linked to one specific serotype. Pulsed-field gel electrophoresis can refine our epidemiologic studies of group B streptococci transmission and acquisition.