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At least 271 records · Page 15Linked to original sources
PubMed1988

DNA sequence and RNA transcription through a site of recombination in a non-neurovirulent herpes simplex virus intertypic recombinant.

RE6 is a herpes simplex type-1 (HSV-1) X herpes simplex type-2 (HSV-2) intertypic recombinant that cannot replicate in the adult mouse nervous system. In the accompanying report, we have shown that HSV-1 sequences between 0.698 and 0.721 map units can restore a partial neurovirulent phenotype to RE6. In this report, we have used comparative DNA sequence analysis of RE6, 17syn+ (HSV-1) and HG52 (HSV-2) to demonstrate that this region contains a site of recombination between HSV-1 and HSV-2 sequences in RE6. High resolution transcription analysis has demonstrated that three readily detected transcripts are present in this region of the genome. In addition, the 5' end of a low abundance 5 kb transcript was also located in the right-hand portion of this region. All the transcripts encoded by HSV-1 and HSV-2 in this region of the genome are expressed by the RE6 recombinant. This and our sequence data suggest that the lack of neurovirulence in RE6 is not due to a simple loss in the expression of a transcript or to a defect in a protein encoded by a gene at the site of recombination.

Base Sequence↗
PubMed1992

Effect of the levels of dissolved oxygen on the expression of recombinant proteins in four recombinant Escherichia coli strains.

Four recombinant strains of Escherichia coli were examined for the effects of the dissolved oxygen level on the level of biomass, the plasmid content, and the level of recombinant protein at the stationary phase of batch growth. Strains JM101/pYEJ001, and TB-1/pYEJ001 (encoding chloramphenicol acetyltransferase), and strain TB-1/p1034, and TB-1/pUC19 (encoding beta-galactosidase) were grown at the constant dissolved oxygen levels of 0, 50, and 100% air saturation, as well as in the absence of dissolved oxygen control. The biomass of all strains under constant aerobic conditions was 12-36 times higher than that under anaerobic conditions, but was the same as or slightly higher than that without dissolved oxygen control. The plasmid content in all strains under aerobic conditions was 2.9-11.7 times higher than that under aerobic conditions. The optimal dissolved oxygen concentration for the specific activity of recombinant proteins was dependent upon the strain. In no strain were constant aerobic conditions optimal. However, because of the effect on biomass, controlled aerobic conditions were optimal for the volumetric activity of recombinant protein in all but one strain.

Aerobiosis↗
PubMed1988

Tumorigenic poxviruses: fine analysis of the recombination junctions in malignant rabbit fibroma virus, a recombinant between Shope fibroma virus and myxoma virus.

Malignant rabbit fibroma virus (MRV) has been shown to be a lethal tumorigenic poxvirus of rabbits derived from a recombination event between Shope fibroma virus (SFV), which induces benign fibromas in rabbits, and myxoma virus, the agent of myxomatosis. We have cloned and sequenced all of the MRV recombination junctions, which are located near the left and right terminal inverted repeat (TIR) regions, and present a composite map of the MRV genome with respect to the relevant gene products. The two junctions closet to the MRV termini, at identical positions at the left and right ends, are at nucleotide 5272 and result in an in-frame fusion protein (ORF T-5) in which the N-terminal 232 aa are derived from an SFV sequence linked to a C-terminus derived from myxoma. At the left MRV TIR the recombination junction distal from the terminus maps to nucleotide 9946 but leaves the adjacent gene virtually unchanged from its SFV homolog. At the right terminus, the relevant junction sequences from MRV and myxoma could not be cloned in wild-type Escherichia coli but were maintained stably in a recA recBC sbcB host. The SFV/myxoma junction at this location maps 5' to a growth factor gene (SFGF) which is related to those encoding epidermal growth factor and transforming growth factor-alpha. As a result, the myxoma growth factor gene has been deleted in MRV and replaced in toto by the SFV gene. The recombination junction upstream from the SFGF gene creates an in-frame fusion in ORF T11-R in which the N-terminal amino acids are derived from myxoma and the remainder from SFV. In summary, MRV has received the following ORFs from SFV: at the left terminus T5 (fusion), T6, T7, and T8; at the right terminus, T5 (fusion), T6, T7, T8, T9-R, SFGF, and T11-R (fusion).

Animals↗
PubMed1994

Potential therapeutic recombinant proteins comprised of peptides containing recombined T cell epitopes.

The complete primary structure of Fel d I2 has been determined and shown to be comprised of two separate polypeptide chains (designated chain 1 and 2). Overlapping peptides covering the entire sequence of both chains of Fel d I have been used to map the major areas of human T cell reactivity. The present study describes three non-contiguous T cell reactive regions of < 30 aa in length that were assembled in all six possible configurations using PCR and recombinant DNA methods. These six recombinant proteins comprised of defined non-contiguous T cell epitope regions artificially combined into single polypeptide chains have been expressed in E. coli, highly purified, and examined for their ability to bind to human cat-allergic IgE and for human T cell reactivity. Several of these recombined T cell epitope-containing polypeptides exhibit markedly reduced IgE binding as compared to the native Fel d I. Importantly, the human T cell reactivity to individual T cell epitope-containing regions is maintained even though each was placed in an unnatural position as compared to the native molecule. In addition, T cell responses to potential junctional epitopes were not detected. It was also demonstrated in mice that s.c. injection of T cell epitope-containing polypeptides inhibits the T cell response to the individual peptides upon subsequent challenge in vitro. Thus, these recombined T cell epitope-containing polypeptides, which harbor multiple T cell reactive regions but have significantly reduced reactivity with allergic human IgE, constitute a novel potential approach for desensitization to important allergens.

Allergens↗
PubMed1985

Characteristics of a swine recombinant influenza virus isolated in 1980: recombination between swine and the earliest Hong Kong (H3N2) viruses.

A recombinant (H1N2, formerly Hsw1N2), A/swine/Ehime/1/80 was found to possess antigenic biological and genomic characteristics different from those of a previous A/swine/Kanagawa/2/78 (H1N2) strain. Five monoclonal antibodies to A/NJ/8/76 differentiated the haemagglutinin molecules of the former virus from the latter, showing that these viruses differed at two-antigenic determinants at least. Immuno-double diffusion tests with antisera to the isolated neuraminidase and neuraminidase-inhibition tests with monoclonal antibodies to different H2N2 and H3N2 viruses revealed that A/swine/Ehime/1/80 strain contained a neuraminidase subunit very similar to that of late human Asian (H2N2) and the earliest Hong Kong (H3N2) viruses. RNA analysis by oligonucleotide mapping suggested that A/swine/Ehime/1/80 may be a recombinant between A/swine/Shizuoka/1/78-like and A/Aichi/2/68 (H3N2)-like viruses. To determine further the gene constellation of this recombinant virus, DNA-RNA hybridizations were performed using DNA segments complementary for swine (H1N1) virus RNA and the entire RNA of three viruses. The molecular hybridization could define the genomic composition of the recombinant, indicating that only the neuraminidase gene of this virus is derived from the earliest Hong Kong (H3N2)-like virus and remaining seven genes are derived from swine (H1N1) virus.

Animals↗
PubMed1995

Recombinant lys-plasminogen, but not glu-plasminogen, improves recombinant tissue-type plasminogen activator-induced coronary thrombolysis in dogs.

OBJECTIVES: This study examined the modification of recombinant tissue-type plasminogen activator (rt-PA)-induced thrombolysis by recombinant lys-plasminogen. BACKGROUND: Recombinant tissue-type plasminogen activator restores flow in the thrombosed coronary artery, but the artery often reoccludes. The rt-PA-induced thrombolysis is a result of activation of plasminogen bound to fibrin in the thrombus and results in generation of the fibrinolytic enzyme plasmin. Small amounts of lys-plasminogen are formed when rt-PA is used. Lys-plasminogen binds to fibrin with a 10-fold greater affinity than the predominant native glu-plasminogen, leading to a loose fibrin structure. METHODS: Dogs with electrically induced occlusive intracoronary thrombus were treated with saline solution (n = 9), glu-plasminogen (2 mg/kg body weight, n = 5) or lys-plasminogen (2 mg/kg, n = 5), followed by infusion of rt-PA (1 mg/kg over 20 min) 10 min later. RESULTS: Reperfusion rates were similar in all groups of dogs, but the time to reflow was lowest in dogs given lys-plasminogen compared with those given saline solution or glu-plasminogen before rt-PA (mean [+/- SE] 14 +/- 2 vs. 22 +/- 2 and 23 +/- 3 min, respectively, p < 0.05). None of the reperfused coronary arteries reoccluded in the lys-plasminogen plus rt-PA group, whereas 75% reoccluded in dogs given saline solution plus rt-PA, and 50% reoccluded in those given glu-plasminogen plus rt-PA. Accordingly, duration of reflow was greater in the lys-plasminogen plus rt-PA group (> 120 vs. 39 +/- 7 and 82 +/- 21 min, respectively, p < 0.05). Plasminogen activator inhibitor-1 activity decreased during rt-PA infusion and thereafter increased in all dogs, but less so in dogs given lys-plasminogen (p < 0.05 vs. those given saline solution before rt-PA). CONCLUSIONS: Treatment with recombinant lys-plasminogen before rt-PA reduces time to reflow and sustains reflow after thrombolysis, whereas glu-plasminogen has no such effect.

Animals↗
PubMedDate not supplied

Expression and characterization of recombinant mouse beta 2-microglobulin type a in insect cells infected with recombinant baculoviruses.

The murine beta 2-microglobulina cDNA was cloned into pAc373 and pVL941 transfer vectors and introduced via homologous recombination into the genome of Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. Both types of recombinant baculoviruses were isolated and used to infect Spodoptera frugiperda (Sf9) lepidopteran cells. beta 2m was synthesized at a substantially higher rate in cells infected with the pVL941-derived virus than when the pAc373-based virus was used. beta 2m was secreted into the culture medium where it accumulated and, under the best conditions, reached an approximate level of 10 micrograms/10(6) cells. Pulse-chase experiments after metabolic labelling with 35S-methionine followed by immunoprecipitation showed that beta 2m was stable, but that the secretion process in infected cells was relatively slow. Recombinant beta 2m was endowed with biological activity and was indistinguishable from that produced by mouse cells in 2D gel analysis. beta 2m was purified to near homogeneity from serum-free culture medium conditioned by recombinant baculovirus-infected cells by using an immunoaffinity column. The use of the insect cell/baculovirus expression system should constitute a suitable source of mouse beta 2m and should aid experiments aimed at unraveling its interactions with mouse class I histocompatibility molecules.

Animals↗
PubMed2006

Roles of nonhomologous DNA end joining, V(D)J recombination, and class switch recombination in chromosomal translocations.

When a single double-strand break arises in the genome, nonhomologous DNA end joining (NHEJ) is a major pathway for its repair. When double-strand breaks arise at two nonhomologous sites in the genome, NHEJ also appears to be a major pathway by which the translocated ends are joined. The mechanism of NHEJ is briefly summarized, and alternative enzymes are also discussed. V(D)J recombination and class switch recombination are specialized processes designed to create double-strand DNA breaks at specific locations in the genomes of lymphoid cells. Sporadic Burkitt's lymphoma and myelomas can arise due to translocation of the c-myc gene into the Ig heavy chain locus during class switch recombination. In other lymphoid neoplasms, the RAG complex can create double-strand breaks that result in a translocation. Such RAG-generated breaks occur at very specific nucleotides that are directly adjacent to sequences that resemble canonical heptamer/nonamer sequences characteristic of normal V(D)J recombination. This occurs in some T cell leukemias and lymphomas. The RAG complex also appears capable of recognizing regions for their altered DNA structure rather than their primary sequence, and this may account for the action by RAGs at some chromosomal translocation sites, such as at the bcl-2 major breakpoint region in the follicular lymphomas that arise in B lymphocytes.

Base Sequence↗
PubMed2006

Random strand transfer recombination (RSTR) for homology-independent nucleic acid recombination.

Random strand transfer recombination (RSTR) relies on the ability of viral reverse transcriptases to undergo homology-independent template switches during DNA synthesis. To facilitate strand transfer events single stranded template DNA was hybridized via noncoding complementary sequence stretches which flank the genes. The resulting bulb shaped heteroduplex was used as template for the reverse transcriptase driven DNA polymerization in which template switch events occur spontaneously. Switches were promoted by induced pausing of the viral polymerase at digoxygenin labeled nucleotides. RSTR of homologous genes was demonstrated by the recombination of two genes encoding (R)-hydroxynitrilase isoenzymes from Prunus amygdolus. For non-homologous RSTR we recombined the genes of the Havea brasiliensis (S)-hydroxynitrilase and the estC gene from Burkholderia gladioli. Base-pairing dependent recombination took place spontaneously and at high frequency between genes with low and high sequence homology.

Base Sequence↗
PubMed2000

The scid recombination-inducible cell line: a model to study DNA-PK-independent V(D)J recombination.

To investigate the molecular mechanisms of the variable (diversity) joining (V(D)J) recombination process at an endogenous gene locus, recombination-inducible cell lines were made from both bcl-2-bearing severe combined immune deficiency (scid) homozygous and scid heterozyous (s/ + ) mice by transforming pre-B cells with the temperature-sensitive Abelson murine leukemia virus (ts-Ab-MLV). These transformants can be induced to undergo immunoglobulin light-chain gene rearrangements by incubating them at the non-permissive temperature. In the case of transformed scid cells, a significant amount of hairpin coding ends are accumulated during recombination induction, but few coding joints are generated. After being shifted to the permissive temperature. however, these cells are capable of opening hairpin ends and forming coding joints. Thus, ts-Ab-MLV transformed scid cells can be readily manipulated for both recombination cleavage and end resolution. However, unlike the rapid coding joint formation in s/ + cells that have the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the process for resolving coding ends in scid cells is slow and error prone, and also appears to be correlated with a reduction in the RAG1/2 expression. Apparently, this process is mediated by a DNA-PK-independent pathway. The fact that the activity of this pathway can be manipulated in vitro makes it possible to delineate the mechanisms in end opening, processing and joining. Therefore, these ts-Ab-MLV transformed scid cell lines offer a model to study the molecular nature as well as the regulation of the DNA-PK-independent pathway in coding end resolution.

Animals↗
PubMed1999

Temperature-induced production of recombinant human insulin in high-cell density cultures of recombinant Escherichia coli.

The construction of expression vectors encoding either the human insulin A- or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported. Using this two-chain approach we also describe the separate isolation of the insulin A- and B-chains from inclusion bodies and their subsequent assembly into native human insulin. The production of the insulin fusion proteins were carried out in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source. The expression of the recombinant genes by temperature-shift in high-cell density cultures of recombinant E. coli resulted in product yields of grams per litre of culture broth, e.g. 4.5 g of insulin B-chain fusion protein per litre of culture broth. This translates into an expression yield of about 800 mg of the insulin B-chain per litre of culture. Under similar cultivation conditions the expression yield of the insulin A-chain corresponds to approximately 600 mg per litre of culture. The metabolic burden imposed on the recombinant cells during temperature-induced production of insulin fusion proteins in high-cell density cultures is reflected in an increased respiratory activity and a reduction of the biomass yield coefficient with respect to glucose.

Amino Acid Sequence↗
PubMed1986

The effects in vivo of purified preparations of murine macrophage colony stimulating factor-1, recombinant murine granulocyte-macrophage colony stimulating factor and natural and recombinant murine interleukin 3 without and with pretreatment of mice with purified iron-saturated human lactoferrin.

The influence of purified natural colony stimulating factor-1 (CSF-1), purified recombinant granulocyte-macrophage (GM)-CSF, purified recombinant interleukin 3 (IL3) and natural IL3 were assessed in mice that were untreated or pretreated with purified iron-saturated human lactoferrin (LF) in order to first suppress myelopoiesis in the mice. S1/S1d mice responded to recombinant GM-CSF and recombinant IL3 in a manner similar to the response of their +/+ littermates. These 4 factors increased the cycling status of hematopoietic progenitors in vivo. The effects were more noticeable if myelopoiesis was first decreased by LF. The effects do not appear to be due to endotoxin contamination. It cannot be discerned from these studies whether the effects are direct ones on the progenitor cells or indirect ones mediated through growth-factor releasing accessory cells. It is possible that effects can be both direct and indirect.

Animals↗
PubMed1986

Coronary thrombolysis by intravenous infusion of recombinant single chain urokinase-type plasminogen activator or recombinant urokinase in baboons: effect on regional blood flow, infarct size and hemostasis.

An occlusive thrombus was produced by thrombin-induced coagulation in the left anterior descending coronary artery of 18 open chest baboons. In six control animals, occlusive thrombosis persisting for 4 hours resulted in a large transmural infarct (66 +/- 4% of the perfusion area, mean +/- SEM). In six animals, single chain urokinase-type plasminogen activator, obtained by recombinant deoxyribonucleic acid (DNA) technology, was infused intravenously at a rate of 20 micrograms/kg per min for 60 minutes after approximately 45 minutes of coronary thrombosis. Persistent reperfusion occurred within 21 +/- 4 minutes (mean +/- SD). The mean duration of occlusion before reperfusion was 72 +/- 6 minutes. Recanalization resulted in a reduction of infarct size (42 +/- 4%, p less than 0.01 versus control animals). Myocardial blood flow in the perfusion area of the left anterior descending coronary artery was 107% of normal 2.5 hours after recanalization. The infusion of recombinant single chain urokinase-type plasminogen activator was not associated with systemic activation of the fibrinolytic system, fibrinogen breakdown or evident bleeding. In six baboons recombinant low molecular weight urokinase (molecular weight 33,000) was infused intravenously at a rate of 20 micrograms/kg per min for 60 minutes after approximately 45 minutes of coronary thrombosis. Persistent reperfusion occurred within 14 +/- 5 minutes (p less than 0.05 versus recombinant single chain urokinase-type plasminogen activator). The mean duration of occlusion was 69 +/- 14 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Selective leakage of host-cell proteins during high-cell-density cultivation of recombinant and non-recombinant Escherichia coli.

Significant leakage of host-cell proteins into the culture medium occurred during high-cell-density cultivation of E. coli. Identification of these medium proteins revealed almost exclusively a periplasmic origin. Release of periplasmic proteins into the culture medium was observed throughout the entire cultivation of recombinant or non-recombinant cells. Leakage was intensified, however, in the final part of high-cell-density cultures (>100 g L(-)(1) dry cell mass) or when a temperature upshift was used for induction of recombinant protein synthesis. After temperature upshift, formation rates and residual cellular concentrations of periplasmic proteins declined with individual rates; e.g., the cellular content of the large periplasmic dipeptide binding protein DppA (57.4 kDa) started to decline about 4 h after the temperature upshift, whereas the smaller periplasmic d-galactose/d-glucose binding protein MglB (33.4 kDa) was already lost during the first hour after the upshift. In addition to periplasmic proteins, the osmotic-shock-sensitive heat-shock protein DnaK was found in significantly higher proportion in the cell-free medium of the temperature-challenged culture than other cytoplasmic proteins. Cell lysis was not observed even after prolonged cultivation. Thus, loss of a subset of cellular proteins of mainly periplasmic origin ordinarily occurs during cultivation and is intensified through stressful conditions in high-cell-density cultures. The selective release of cellular proteins of periplasmic origin offers the opportunity to simplify the downstream processing of recombinant proteins directed to the periplasm of E. coli.

Bioreactors↗
PubMed2001

The effect of exogenous luteinizing hormone (LH) on oocyte viability: evidence from a comparative study using recombinant human follicle-stimulating hormone (FSH) alone or in combination with recombinant LH for ovarian stimulation in pituitary-suppressed women undergoing assisted reproduction.

PURPOSE: The purpose of this prospective, randomized study was to compare ovarian response and oocyte and embryo yields in women undergoing ovulation induction for IVF/ICSI using recombinant human FSH (rhFSH) alone or in combination with recombinant human LH (rhLH). METHODS: Patients were randomized to receive rhFSH alone (group F; n = 13) or rhFSH + rhLH (group L; n = 15). rhFSH was administered according to a step-down protocol; patients assigned to group L received rhLH at a fixed dose of 75 IU (1 ampoule) throughout the treatment period. RESULTS: The total dose of rhFSH, number of growing follicles, and serum concentrations of estradiol (E2) on the day of hCG administration were similar in both treatment groups. However, the percentage of metaphase II oocytes and fertilization rate were significantly higher in group F than in group L. The lower fertilization rates associated with rhLH were also seen in a subgroup of patients from group L who had undergone a previous ART cycle stimulated with FSH only and thus acted as their own controls. However, when in vitro fertilization (IVF) and intracytoplasmic sperm injection cycles were considered separately, differences in fertilization rates were statistically significant only for oocytes treated by conventional IVF. CONCLUSIONS: This study shows that the addition of recombinant LH to recombinant FSH in pituitary-suppressed women undergoing ART does not improve the ovarian response and even may have a negative impact on oocyte maturation and fertilization.

Adult↗
PubMed2000

Recombination signal sequences restrict chromosomal V(D)J recombination beyond the 12/23 rule.

The genes encoding the variable regions of lymphocyte antigen receptors are assembled from variable (V), diversity (D) and joining (J) gene segments. V(D)J recombination is initiated by the recombinase activating gene (RAG)-1 and -2 proteins, which introduce DNA double-strand breaks between the V, D and J segments and their flanking recombination signal sequences (RSSs). Generally expressed DNA repair proteins then carry out the joining reaction. The conserved heptamer and nonamer sequences of the RSSs are separated by non-conserved spacers of 12 or 23 base pairs (forming 12-RSSs and 23-RSSs). The 12/23 rule, which is mediated at the level of RAG-1/2 recognition and cutting, specifies that V(D)J recombination occurs only between a gene segment flanked by a 12-RSS and one flanked by a 23-RSS. Vbeta segments are appended to DJbeta rearrangements, with little or no direct Vbeta to Jbeta joining, despite 12/23 compatibility of Vbeta 23-RSSs and Jbeta12-RSSs. Here we use embryonic stem cells and mice with a modified T-cell receptor (TCR)beta locus containing only one Dbeta (Dbeta1) gene segment and one Jbeta (Jbeta1) gene cluster to show that the 5' Dbeta1 12-RSS, but not the Jbeta1 12-RSSs, targets rearrangement of a diverse Vbeta repertoire. This targeting is precise and position-independent. This additional restriction on V(D)J recombination has important implications for the regulation of variable region gene assembly and repertoire development.

Alleles↗
PubMed1976

On the mechanism of genetic recombination: electron microscopic observation of recombination intermediates.

This paper deals with the nature of recombination intermediates. Using the electron microscope to study the DNA of the plasmid colicin E1, we have observed more than 800 molecules that appear to represent intermediates in the process of recombination. Specifically, after isolating colicin DNA and linearizing it with the restriction enzyme EcoRI, we find crossed molecules with twice the normal colicin DNA content. These forms consist of two genome-length elements held together at a region of DNA homology. The molecules can be recovered from wild type and Rec B-C host cells but are not present among the colicin DNA forms isolated from recombination-deficient Rec A cells. We have termed the experimentally observed molecules "chi forms" and believe that they represent the recombination intermediate of the Holliday model.

Colicins↗
PubMed1977

On the mechanism of genetic recombination: the maturation of recombination intermediates.

DNA molecules of the plasmid ColEl are normally recovered from wild-type cells as a set of monomer- and multimer-size rings. The data of this paper show that the multimer-size species are a product of genetic recombination. Multimer rings do not arise after transfection of purified monomers into bacterial host cells lacking a functional recA recombination system. Analogously, purified dimers, trimers, and tetramers, transfected into recA- cells, can replicate, but are constrained to remain in those conformations. Only upon transfection into rec+ cells can they regenerate the full spectrum of monomer- and multimer-size species. In this paper we trace the flow of genetic information from the monomer to the multimer state and back again under the guidance of the recA recombination system. The formation of multimer-size DNA rings is discussed as a natural consequence of the maturation of a Holliday recombination intermediate formed between two monomer plasmid genomes.

Chromosomes↗